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

Chiral discrimination of thyroxine (T4) 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 T4 under the optimized conditions. The calibration curves of both D- and L-T4 showed an excellent linearity with coefficient of determination (R2) > 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.


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][2][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, is secreted by the thyroid gland into the circulatory system [4][5][6][7][8][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][9][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][10][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 Jisun Lee and Suraj Adhikari contributed equally to this work.
* Wonjae Lee wlee@chosun.ac.kr 1 3 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][14][15][16]. In crown ether-type CSPs, the complexes formed between protonated amino acids under acidic conditions, and chiral crown ether 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 highperformance 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.

Liquid Chromatography and Tandem Mass Spectrometry
The UHPLC-MS/MS system comprised 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 Nexera X2 LC-30AD quaternary pump, a column oven (CTO-20AC) and a degasser (DGU-20A5R). The MS system consisted of an electrospray ionization interface and a triple quadrupole (QqQ) mass analyzer.

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. Afterward, 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. Methanol was used as blank during the preparation.

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 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.

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 were 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 were 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 were 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 Although TFA showed shortest retention of T 4 enantiomers, the worst resolution and MS signal intensity (Fig. 1D) of T 4 enantiomers compared to those of FA ( Fig. 1A-C) was observed. Overall, the lowest concentration of FA in the mobile phase showed the highest MS intensity with improved the peak shape (Fig. 1A) and thus, the best results in this study were achieved using 60% methanol/water (v/v) with 0.1% FA. Under the optimum separation condition, this developed analytical method was carried out in plasma. Figure 2 illustrates the three MRM chromatograms of blank plasma, internal standard (IS, L-phenylglycine, 1 μg mL −1 ) spiked to healthy plasma and standard solution of DL-T 4 (20 μg mL −1 ) spiked to plasma with IS at 40 °C optimized column temperature. The chromatogram ( Fig. 2A) of the blank plasma showed no significant matrix interference peaks. In plasma containing standard solutions of DL-T 4 (20 μg mL −1 ), the T 4 enantiomers were separated on the baseline and each enantiomer peak can be ascertained with the retention of standard D-and L-T 4 (Fig. 2C).

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 to 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 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 [Eqs. (1) and (2)] as: 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 Fig. 3A. For ln α vs 1/T, the plot that characterize the separation was linear (Fig. 3B) 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 (Δi°) (− 1.77 kJ mol −1 , Table 3) in our study predicted the efficient binding between analyte and CSP [25].

Linearity and Sensitivity
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. 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 = 0.1587 × -0.1237 and y = 0.0784 × -0.0646, respectively. The high R 2 value of both enantiomers (> 0.9997) indicated that there was good linearity and shown in Fig. 4. 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 bioanalytical 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. 5.
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. 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.
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, experimental design, editing and supervision were contributed by W. Lee. Analysis tools, financial support, investigation and supervision were provided by H.-R.Yoon.
Funding Health Fellowship Foundation, 2021

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