General screening and optimization strategy for fast chiral separations in modern supercritical fluid chromatography

Analytica Chimica Acta 950 (2017) 199e210

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Analytica Chimica Acta journal homepage: www.elsevier.com/locate/aca

General screening and optimization strategy for fast chiral separations in modern supercritical fluid chromatography kova  a, *, Michal Dousa b Lucie Nova a b

lov Department of Analytical Chemistry, Faculty of Pharmacy, Charles University in Prague, Heyrovsk eho 1203, 500 05 Hradec Kra e, Czechia Zentiva, k. s. A Sanofi Company, Praha, U Kabelovny 130, 102 37 Praha 10, Czechia

h i g h l i g h t s

g r a p h i c a l a b s t r a c t


General screening approach for fast SFC chiral separation was developed.
The best enantioselectivity in SFC was obtained with tris(3,5dimethylphenylcarbamate) substituted stationary phases based on both amylose and cellulose.
Combined additive composed of 0.1% DEA and 0.1% TFA provided the best enantioselectivity for generic screening.
Volatile buffers were found to be a viable option for SFC chiral screening.
Tetrahydrofuran was the most convenient injection solvent for chiral SFC analyses.

a r t i c l e i n f o

a b s t r a c t

Article history: Received 2 September 2016 Received in revised form 22 October 2016 Accepted 2 November 2016 Available online 21 November 2016

High throughput general chiral screening method using supercritical fluid chromatography was developed. This method takes an advantage of very fast gradient screening (3 min þ 1 min isocratic hold) and generic enantioselectivity of the combined additive formed by 0.1% trifluoroacetic (TFA) acid and 0.1% diethylamine (DEA). The TFA/DEA combined additive was systematically added to organic modifiers methanol and isopropanol. Among five tested polysaccharide-based chiral stationary phases, amylose tris(3,5-dimethylphenylcarbamate) and cellulose tris(3,5-dimethylphenylcarbamate) provided the best enantioseparation success rate. Therefore, the proposed initial first-line screening includes four experiments using these two stationary phases and the above mentioned two combinations: CO2/methanol and CO2/isopropanol þ the combined additive. If these stationary phases fail in the screening step, cellulose tris(3-chloro-4-methylphenylcarbamate) and cellulose tris(3,5-dichlorophenylcarbamate) can be proposed for the screening in the second line. For further optimization in case of insufficient resolution obtained in the screening phase fine tuning of temperature, BPR pressure and gradient slope was tested with unsuccessful results. An improvement of enantioselectivity was obtained only when gradient elution was replaced by isocratic elution with substantially lower amount of organic modifier, when changing the concentration of the additive or when using combined organic modifier, such as methanol/acetonitrile (1:1). Finally, to enable the MS compatibility, also volatile additives including ammonium formate and ammonium acetate were tested.

Keywords: Supercritical fluid chromatography SFC Chiral stationary phases Organic modifier Combined additive Enantioselectivity Pharmaceuticals

* Corresponding author. E-mail address: [email protected] (L. Nov akov a). http://dx.doi.org/10.1016/j.aca.2016.11.002 0003-2670/© 2016 Elsevier B.V. All rights reserved.

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The results were more encouraging than expected. Volatile buffers thus make an interesting option in chiral SFC screening methods, however, at the cost of somewhat lower enantioselectivity. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Supercritical fluid chromatography (SFC) has already become quite well-established in separation of chiral compounds [1e10]. However, it is not yet considered a first-choice option for routine quality control (QC) in pharmaceutical industry, although it meets the criteria of high efficiency and fast separation, which are required due to the huge number of samples to be analyzed. The reasons for a lower popularity of SFC in routine QC originates in the past drawbacks of the technique, such as much lower method robustness, repeatability and sensitivity of available instrumentation. Therefore, HPLC in both reversed phase and normal phase modes are still considered to be the methods of the first choice [11e13] with the current trends of ultra-fast enantioseparations using superficially porous particles [14e16] or teicoplanin sub-2 mm totally porous silica particles [17] stationary phases providing separations within seconds [18]. Recently, new SFC platforms and new stationary phases have been commercially introduced in order to extend application potential and reliability of SFC method and to compensate for the above stated drawbacks [19,20]. The generic screening should consist of limited number of fast chromatographic experiments, which can be applied to various racemates and be helpful in quick finding of conditions for successful separation. The state of the art in screening chiral SFC strategies employs several polysaccharide based chiral stationary phases and CO2/methanol mobile phase with the addition of trifluoroacetic acid (TFA) for the analysis of acidic compounds and isopropylamine (IPA) or another amine for basic and neutral compounds [1,3,21e23]. Isocratic elution using 10 and/or 20% of alcoholic organic modifiers (typically methanol and/or isopropanol) has been still preferred even in several quite recent modern methods for chiral screening despite very long retention times for some compounds and difficulty of prediction and set-up of analysis times [21,23e25]. In this regard the method development remains substantially less effective and still time-consuming. Modern screening approaches request for very fast analyses, even in chiral analysis. Therefore, a quick gradient elution is much more convenient for fast screening purposes. Similarly to isocratic methods, gradient screening approaches were using alcoholic modifiers and various additives, such as TFA, IPA, diethylamine (DEA) or thiethylamine (TEA) for the improvement of the peak shapes [22,26e28]. However, in many cases these were still very time consuming requiring about 15e25 min analysis times [22,26,27]. Until recently, gradient approaches were less commonly used, probably due to instrumentation constraints that did not enable sufficient method reproducibility. Few rapid methods using 2.5 min gradient method with CO2/methanol mobile phases and 0.1% TEA [28], methanol, ethanol or isopropanol with the addition of NH4OH [29] and CO2/ methanol with 25 mM isobutylamine [3] as the additives have been published. The latter employed a combination of 3 min isocratic and 6 min gradient elution. However, considering the basic nature of additives, these approaches may be considered less generic. The chiral screening method speed and efficiency may be further increased also using multi-column parallel approaches [30,31]. Currently available commercial SFC systems enable using up to 8 columns in parallel. An important advance in the screening approaches in SFC

included the application of a combined additive, i. e. utilization of IPA and TFA simultaneously in one mobile phase [32e36]. Due to this approach there was no need for compound classification any more. Moreover, an improved separation efficiency and enatioselectivity has been reported, especially for zwitterionic compounds. However, a combined additive (0.5% TFA þ 0.5% IPA) present in methanol/CO2 mobile phases resulted in problems with the system stability in this study due to the salt complex formation [33]. Alternatively, also a combination of TFA/TEA (0.2e0.3%) was reported to provide excellent enantioselectivity [37]. Unfortunately, use of isocratic elution significantly decreased the throughput of these reported approaches [24,25,32e34]. The aim of this work was to develop a quick general screening and optimization strategy for fast SFC separation of chiral pharmaceuticals using modern approaches to SFC chiral screening in one design. SFC method development was performed using polysaccharide based chiral columns packed with 3 mm particles and new ultra-high performance supercritical fluid chromatography (UHPSFC) instrumentation platform. A procedure for fast and straightforward method development was designed for separation of chiral basic, neutral and acidic compounds from the group of APIs (active pharmaceutical ingredients) and their intermediates using combined acidic/basic additive added to the organic modifiers. The influence of individual variables on enantioresolution, analysis time and peak asymmetry was evaluated. Finally, the feasibility of use of volatile MS friendly additives in chiral screening and the influence of injection solvent were evaluated as well. 2. Experimental 2.1. Chemicals and reagents Reference standards of pharmaceuticals including neutrals and bases (alaptide, aprepitant intermediate, atomoxetine, bocsaxagliptin, cinacalcet, darifenacin, epinephrine, ezetimibe, fesoterodine, fesoterodine intermediate, indacaterol, maraviroc, maraviroc intermediate ester, tamsulosin, tolterodine, zolmitriptan) and acids (ambrisentan, cetirizin, ibuprofen, ketoprofen) were provided by Zentiva a.s., (Prague, Czech Republic) and Sigma Aldrich (Prague, Czech Republic), respectively. Except for aprepitant, epinephrine and indacaterol which were at the disposal only in a racemate form, all pharmaceuticals were available in both R and S configuration, for boc-saxagliptin in S,S,S,S and R,R,R,R and for ezetimibe in R,S,S and S,R,R respectively. Methanol, ethanol, isopropanol, acetonitrile, heptane HPLC gradient grade and tetrahydrofuran HPLC grade were provided by Sigma Aldrich (Prague, Czech Republic). Mobile phase additives including diethylamine (99.5%), trifluoroacetic acid (99%), ammonium acetate (>99.99%) and ammonium formate (>98%) were purchased from Sigma Aldrich (Prague, Czech Republic). 2.2. Ultra-high performance supercritical fluid chromatography The supercritical fluid chromatography system Acquity UPC2 (Waters, Prague, Czech Republic) consisted of Acquity UPC2 binary solvent manager, Acquity UPC2-FL sample manager, Acquity UPC2 convergence manager, Acquity column manager and Acquity UPC2

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PDA detector. All injected sample solutions were stored in the autosampler at 4  C. The partial loop with needle overfill mode was set up to inject 10 mL. Methanol was used as a needle wash solvent. Gradient elution was performed using CO2 (>99.995%, LindeGas, Hradec , Czech Republic) and various combinations of modifiers Kr alove (methanol, ethanol, isopropanol and acetonitrile) and additives (trifluoroacetic acid, diethylamine, ammonium acetate, ammonium formate) at a flow-rate of 3.0 mL/min. Gradient program started at 5% of organic modifier and was linearly increased up to 30% in 3 min. An isocratic step was kept at 30% of organic modifier for 1 min followed by column equilibration (2 min). The influence of temperature was evaluated in the range of 10e60  C. The BPR (back-pressure regulator) pressure was set to 1500 psi and its variations were evaluated in the range of 1500e2500 psi. UV detection by means of PDA detector was performed at extracted wavelength corresponding to the absorption maximum of individual tested compounds. The separation was performed using polysaccharide based chiral stationary phases including: Lux 3u Amylose-2: coated amylose tris(5-chloro-2-methylphenylcarbamate), provided by Phenomenex (Chromservis, Prague, Czech Republic), Kromasil 3-Amycoat: coated amylose tris(3,5-dimethylphenylcarbamate), provided by Kromasil (Chromservis, Prague, Czech Republic), Lux 3u Cellulose1: coated cellulose tris(3,5-dimethylphenylcarbamate), provided by Phenomenex (Chromservis, Prague, Czech Republic), Lux 3u Cellulose-2: coated cellulose tris(3-chloro-4methylphenylcarbamate), provided by Phenomenex (Chromservis, Prague, Czech Republic) and Chiralpak IC-3: immobilized cellulose tris(3,5-dichlorophenylcarbamate), provided by Daicel (Illkirch Cedex, France). All columns in this study had dimensions of 150  4.6 mm and 3 mm particles. 2.3. Standard solutions The stock standard solutions of the pharmaceuticals were prepared in tetrahydrofuran due to its good compatibility with SFC mobile phases and overall good solubility for the selected set of compounds. Less soluble compounds including alaptide, atomoxetine, darifenacin, epinephrine, indacaterol and cetirizine were dissolved in a mixture of tetrahydrofuran/methanol (9:1). Tamsulosin was not soluble at all in tetrahydrofuran, therefore the stock

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solution was prepared in methanol. The concentrations of the stock solutions were 1 mg/mL. Stock solutions were further diluted with tetrahydrofuran in order to obtain a mixture of both enantiomers at a concentration of 0.1 mg/mL, which was used for the injection into SFC system for SST measurements. For screening purposes, a concentration of 0.3 mg/mL was used for an active isomer for faster and easier identification of the isomers. In case of doubts individual injections of each isomer were performed to confirm the peak identity. 3. Results and discussion 3.1. Selection of stationary and mobile phases for fast SFC screening Selection of correct stationary phase for chiral separation still remains challenging both in LC and SFC techniques. Based on research previously performed in SFC and LC screening approaches [11e13,21e25,38] and based on practical experience with chiral separations, five the most promising stationary phases were selected for the initial screening and subsequent comparison. These included cellulose tris(3,5-dimethylphenylcarbamate), amylose tris(3,5-dimethylphenylcarbamate) and chlorinated phases including cellulose tris(3-chloro-4-methylphenylcarbamate), amylose tris(5-chloro-2-methylphenylcarbamate) and finally cellulose tris(3,5-dichlorophenylcarbamate). The group of analytes was selected in order to reflect the current state at the drug market including acidic, basic and neutral analytes and being both APIs and pharmaceutical intermediates that have to be subjected to chiral purity control. Table 1 shows basic physico-chemical properties of the tested chiral compounds. Log P values reveal, that the range of polarity was quite large (0.06e6.19). Most of the tested compounds had basic character which also corresponds to the typical situation at the drug market. Following the state-of-the art of SFC separations and in agreement with the previous findings, the first comparison was made using CO2/methanol þ0.1% TFA for the analysis of acidic compounds and CO2/methanol þ0.1% DEA for basic and neutral compounds. Two comparative mobile phases were made by a combination of 0.1% DEA and 0.1% TFA added simultaneously first to methanol/CO2 and secondly to isopropanol/CO2. In contrast to previously described technical problems with the system stability described in Ref. [33] when using a combination of 0.5% TFA and 0.5% IPA in methanol/CO2, similar issues have never been observed through

Table 1 Physico-chemical properties of chiral pharmaceuticals used in this study. Compound

Molecular formula Molecular weight (g/mol) log P

Alaptide Ambrisentan Aprepitant int Atomoxetin Boc-saxagliptin Cetirizine Cinacalcet Darifenacin Epinephrine Ezetimibe Fesoterodine Fesoterodine int Ibuprofen Indacaterol Ketoprofen Maraviroc Maraviroc int ester Tamsulosin Tolterodine Zolmitriptan

C9H14N2O2 C22H22N2O4 C21H19F6O3N C17H21NO C19H26N2O2 C21H25ClN2O3 C22H22F3N C28H30N2O2 C9H13NO3 C24H21F2NO3 C26H37NO3 C22H31NO2 C13H18O2 C24H28N2O3 C16H14O3 C29H41F2N5O C9H11NO2 C20H28N2O5S C22H31NO C16H21N3O2

182.22 378.42 447.37 255.35 314.42 388.89 357.41 426.55 183.20 409.43 411.58 341.49 206.29 392.49 254.28 513.67 165.18 408.51 325.49 287.36

0.06 2.82 5.05 3.36 1.12 0.86 6.19 3.79 0.43 3.96 4.25 3.43 3.84 4.09 3.61 5.30 0.93 2.14 5.23 0.46

pKa acidic pKa basic Detection wavelength (nm) Acid-base characteristic Active enantiomer e 0.97 e e 14.74 3.60 e 15.70 9.69 9.72 14.27 9.58 4.85 8.68 3.88 14.80 e 10.08 10.13 12.57

Note: Acid-base characteristic: A eacid, B e base, N eneutral, Z - zwitterionic.

e 2.84 e 10.15 7.58 7.79 9.19 9.32 8.91 0.20 10.60 10.82 e 0.90 e 10.24 6.70 8.78 10.68 9.52

210 220 220 220 215 230 220 220 220 230 220 220 220 260 250 215 215 220 220 220

N A N B B Z B B B N B B A B A B N B B B

S S R R S,S,S,S R R S R R,S,S R R R,S R R,S S S R R S

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the course of this study despite using also higher concentrations of the additives later in the method optimization phase. This may be attributed to a better solubility of TFA-DEA salt complexes compared to TFA-IPA salt. TFA-DEA salt is formed in the ratio 1:1, while in case of TFA-IPA it may be 2:1 leading to worse salt solubility in CO2/methanol mobile phases. All the screening experiments were performed in quick gradient elution (3 min þ 1 min isocratic hold) described in Section 2.2, which substantially increased the throughput of the screening phase compared to the isocratic elution. Longer separation time (6 min þ 1 min isocratic hold) was also tested in the preliminary experiments to verify if the enantioresolution was not compromised due to the short analysis times. The impact of the time on enantioseparation was negligible, thus short gradient time of 3 min was selected. The comparison of the success rate of the enantioseparation using single and combined additives on all five tested stationary phases is shown in Fig. 1. In this presentation completely separated peaks as well as partially separated peaks are taken as “successful separation”, because method optimization is considered as following step, if necessary. Generally, combined TFA/DEA additive in methanol/CO2 (blue column in Fig. 1) provided always at least the same, but generally better results compared to single additives in methanol/CO2 (black column). This improvement could be both improvement of partial separation to complete baseline separation or non-resolved enantiomeric pair obtained in case of single additives while the separation was obtained with the combined TFA/ DEA additive. The combined TFA/DEA additive added to isopropanol/CO2 demonstrated overall lower success rate in the enantioseparation. However, its benefit was different and often complementary enantioselectivity compared to methanol with the combined TFA/DEA additive, especially on Amycoat stationary phase. Overall, the best enantioselectivity was obtained on Amycoat and on Cellulose 1 columns when using CO2/methanol þ0.1% DEA and 0.1% TFA leading to at least partial separation of 60% of enantiomeric pairs in both cases (12 out of 20). The second best enantionselectivity was observed on Amycoat column when using CO2/isopropanol þ0.1% DEA and 0.1% TFA providing separation for 55% of enantiomeric pairs. Interestingly, both stationary phases contained 3,5-dimethylphenylcarbamoyl functional groups, either on amylose or on cellulose. As already mentioned, a great benefit

was the different enantioselectivity provided by the two organic modifiers with the combined TFA/DEA additive, which is shown in Fig. 2 for the two columns providing the best results. Thus, putting the two conditions together provided very good success separation rate (18 enantiomeric pairs out of 20 at least partially resolved using these four conditions). To complete this set of experiments, further analyses were performed also with CO2/ethanol þ0.1% DEA and 0.1% TFA on Amycoat and Cellulose 1 stationary phases to verify, if there was not some interesting enantioselectivity missed. The results showed, that no additional enantioselectivity was observed compared to CO2/methanol and CO2/isopropanol with the combined TFA/DEA additive. The selectivity of this CO2/ethanol-

Fig. 2. Comparison of the success-rate of enantioseparation of individual compounds for Cellulose 1 (blue) and Amycoat (black/gray) stationary phases using CO2/methanol and CO2/isopropanol with the combined TFA/DEA additive. Successful separation (full separations and partial separations) is represented by the columns above the x-axis (þ), while non-successful separation is represented by the columns bellow the x-axis (). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 1. Comparison of the success-rate of enantioseparation of the 20 tested compounds on the five tested polysaccharide-based stationary phases in gradient chiral screening. CO2/ methanol with the addition of 0.1%DEA was used for the analysis of basic and neutral compounds and 0.1% TFA for the analysis of acidic compounds. Comparison was made with CO2/methanol and CO2/isopropanol with a combined additive composed of 0.1% TFA and 0.1% DEA.

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based mobile phase was mostly similar to that of CO2/methanol with the combined TFA/DEA additive. Graphical presentation in Fig. 2 reveals straightforward and challenging separations. There are several compounds (e.g. alaptide, boc-saxagliptin, cinacalcet and few others), for which 3e4 different conditions out of the 4 provided at least partial separation. On the other hand, challenging analytes (e.g. epinephrine, fesoterodine int. and cetirizine) have shown only one successful separation providing less flexibility for further method optimization. There were only two compounds which did not provide any separation at all in the initial screening phase at selected first-line screening conditions e tolterodine (no enantioresolution at all) and ambrisentan (resolved on Cellulose 2). In the light of these results the recommendation for the initial chiral screening in UHPSFC would be a combination of cellulose tris(3,5dimethylphenylcarbamate) and amylose tris(3,5dimethylphenylcarbamate) using CO2/methanol and CO2/isopropanol with the combined additive composed of 0.1% DEA and 0.1% TFA. The third ranked stationary phase was Cellulose 2 (cellulose tris(3-chloro-4-methylphenylcarbamate)) using CO2/isopropanol with the combined TFA/DEA additive and Chiralpak IC3 (cellulose tris(3,5-dichlorophenylcarbamate)) using CO2/methanol with the combined TFA/DEA additive providing the same success rate of separation (45%, 9 pairs out of 20) in the screening phase. These stationary phases are further options for fine-tuning

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enantioselectivity. 3.2. Detailed optimization of SFC chiral methods Effective screening approach enabled by gradient elution and selection of a good combination of stationary and mobile phases enabled to obtain full enantioseparation for some compounds already in the screening phase. In this study strict criteria were defined for the separation of the two enantiomers with the respect to the real-life situation. When the enantiopurity control is performed, typical limit for a chiral impurity is 0.15% relative to API. In the real-life situation this results in very wide peak of API often leading to strong tailing due to the column overload. Therefore, the criteria for resolution (Rs) was set-up to 2.0 rather than commonly accepted 1.5, which would not be sufficient in the above described case if the impurity is eluted right after the API. If the criteria of Rs  2.0 was met, the system suitability test (SST) measurement was performed in order to verify method repeatability (Table 2). If the resolution was lower and also in case of partial separations, where the resolution could not have been calculated, further optimization followed. Complete enantioseparation was obtained for several compounds already during the screening step up to a different extent on the five tested stationary phases. This success rate corresponded to the overall success rate of the columns shown in Fig. 1. Thus, for

Table 2 Final analytical conditions (stationary phase, mobile phase, gradient elution (G) or isocratic elution (ISO)) for both screening phase optimal separations and finely optimized separations of all compounds. Retention times, repeatability and resolution are also shown. tR1 Alaptide

3.224 (R) 3.008 (R) 3.529 (S*) 2.212 (S*) Aprepitant int. 1.184 1.918 Atomoxetine 2.375 (S) 1.204 (S) 3.928 (S) 3.638 (S) Boc-saxagliptin 2.583 (S*SSS) 0.911 (S*SSS) 2.408 (S*SSS) 1.772 (S*SSS) Cinacalcet 2.147 (S) 1.639 (S) 2.385 (R*) 3.080 (R*) Darifenacin 4.716 (R) 2.637 (S*) 3.149 (R) Epinephrine 4.867 Ezetimibe 3.577 (RSS*) 2.261 (RSS*) Fesoterodine 5.875 (R*) Fesoterodine int 6.949 (R*) Indacaterol 3.459 Maraviroc 5.346 (S*) Maraviroc int ester 2.128 (R) 1.916 (R) 2.647 (R) 2.506 (S*) Tamsulosin 8.508 (R*) Tolterodine 11.109 (S) Zolmitriptan 3.940 (S*) 3.717 (R) Ambrisentan 2.615 (S,S*) Cetirizine 3.836 (S) 4.132 (S) Ibuprofen 5.460 (S*) Ketoprofen 8.375 (S*)

tR2

RSD tR1 [%] RSD tR2 [%] RSD A1 [%] RSD A2 [%] RS

Column

Elution

Mobile phase

3.529 (S*) 3.697 (S*) 4.538 (R) 3.474 (R) 1.623 2.269 2.924 (R*) 1.826 (R*) 4.138 (R*) 4.796 (R*) 3.236 (RRRR) 1.312 (RRRR) 2.597 (RRRR) 2.180 (RRRR) 2.682 (R*) 2.806 (R*) 2.530 (S) 3.604 (S) 5.840 (S*) 3.368 (R) 5.030 (S*) 5.960 4.098 (SRR) 3.218 (SRR) 6.792 (S) 8.206 (S) 5.010 6.405 (R) 2.476 (S*) 2.035 (S*) 2.815 (S*) 2.698 (R) 10.094 (S) 12.365 (R) 4.765 (R) 4.668 (S*) 3.201 (R,R) 4.038 (R*) 4.762 (R*) 5.940 (R) 9.109 (R)

0.04 0.04 0.06 0.04 0.08 0.03 0.02 0.06 0.04 0.03 0.02 0.08 0.02 0.04 0.06 0.05 0.02 0.04 0.03 0.10 0.05 0.08 0.09 0.04 0.08 0.07 0.12 0.07 0.05 0.04 0.03 0.03 0.06 0.31 0.03 0.02 0.05 0.01 0.04 0.06 0.12

Amycoat Amycoat Cellulose 2 Cellulose 2 Cellulose 2 Cellulose 2 Cellulose 1 Cellulose 1 Cellulose 2 Cellulose 2 Amycoat Amycoat Cellulose 1 Cellulose 1 Amycoat Amycoat Cellulose 1 Cellulose 1 Cellulose 1 Amycoat Amycoat Cellulose 2 Amycoat Amycoat Cellulose 1 Amycoat Amycoat Cellulose 1 Chiralpak IC3 Cellulose 1 Cellulose 2 Cellulose 2 Amycoat Amycoat Cellulose 2 Amycoat Cellulose 2 Cellulose 1 Cellulose 1 Chiralpak IC3 Chiralpak IC3

G ISO G ISO ISO G G ISO G ISO G ISO G ISO G ISO G ISO ISO ISO ISO ISO G ISO ISO ISO ISO ISO G G G G ISO ISO ISO ISO ISO G ISO ISO ISO

MeOH þ 0.1% TFA þ 0.1% DEA MeOH þ 0.1% TFA þ 0.1% DEA MeOH þ 0.1% TFA þ 0.1% DEA MeOH þ 0.1% TFA þ 0.1% DEA 2-Pr-OH þ 0.1% TFA þ 0.1% DEA 2-Pr-OH þ DEA 0.1% MeOH þ 0.1% TFA þ 0.1% DEA MeOH þ 0.1% TFA þ 0.1% DEA 2-Pr-OH þ 0.1% TFA þ 0.1% DEA 2-Pr-OH þ 0.1% TFA þ 0.1% DEA MeOH þ 0.1% TFA þ 0.1% DEA MeOH þ 0.1% TFA þ 0.1% DEA MeOH þ 0.1% TFA þ 0.1% DEA MeOH þ 0.1% TFA þ 0.1% DEA 2-Pr-OH þ 0.1% TFA þ 0.1% DEA 2-Pr-OH þ 0.1% TFA þ 0.1% DEA MeOH þ 0.1% TFA þ 0.1% DEA MeOH þ 0.1% TFA þ 0.1% DEA 2-Pr-OH þ 0.1% TFA þ 0.1% DEA 2-Pr-OH þ 0.1% TFA þ 0.1% DEA 2-Pr-OH þ 0.1% TFA þ 0.5% DEA MeOH þ 0.1% TFA þ 0.1% DEA MeOH þ 0.1% TFA þ 0.1% DEA MeOH þ 0.1% TFA þ 0.1% DEA 2-Pr-OH þ 0.1% TFA þ 0.1% DEA 2-Pr-OH þ 0.1% TFA þ 0.1% DEA MeOH þ 0.1% TFA þ 0.5% DEA MeOH þ 0.1% TFA þ 0.1% DEA MeOH þ 0.1% TFA þ 0.1% DEA MeOH þ 0.1% TFA þ 0.1% DEA MeOH þ 0.1% DEA MeOH þ 0.1% TFA þ 0.1% DEA 2-Pr-OH þ 0.1% TFA þ 0.1% DEA MeOH þ DEA 0.5% MeOH þ 0.1% TFA þ 0.1% DEA MeOH þ 0.1% TFA þ 0.1% DEA MeOH þ 0.1% TFA þ 0.1% DEA MeOH þ 0.1% TFA þ 0.1% DEA MeOH þ 0.1% TFA þ 0.1% DEA MeOH:ACN (1:1) þ 0.1% TFA þ 0.1% DEA MeOH:ACN (1:1) þ 0.1% TFA þ 0.1% DEA

0.04 0.04 0.05 0.03 0.05 0.02 0.01 0.05 0.03 0.02 0.04 0.04 0.02 0.05 0.05 0.06 0.02 0.05 0.05 0.09 0.08 0.09 0.12 0.03 0.07 0.11 0.18 0.07 0.03 0.02 0.02 0.04 0.10 0.29 0.03 0.04 0.06 0.01 0.04 0.24 0.11

0.86 0.33 0.82 0.29 0.77 0.95 0.15 0.22 0.31 0.32 0.38 0.28 0.22 0.24 0.16 0.24 0.10 0.20 0.41 0.21 0.96 0.23 0.22 0.24 0.42 0.27 0.49 0.37 0.37 0.50 0.59 0.39 0.34 0.56 0.33 0.10 0.32 0.10 0.19 0.30 0.45

0.45 0.49 0.40 0.33 0.32 0.27 0.23 0.21 0.48 0.05 0.14 0.46 0.16 0.79 0.20 0.36 0.12 0.18 0.44 0.17 0.79 0.31 0.43 0.34 0.46 0.45 0.57 0.37 0.29 0.28 0.53 0.65 0.53 0.85 0.52 0.10 0.40 0.31 0.13 0.53 0.43

3.59 3.55 8.97 8.20 3.77 3.86 8.55 4.40 2.14 2.75 4.01 2.22 2.50 2.24 7.60 6.49 2.00 2.75 2.46 2.95 6.55 2.82 4.51 3.90 2.27 2.13 2.86 2.69 5.82 2.36 1.45 2.84 2.33 1.25 2.49 2.51 3.07 2.22 2.30 2.08 2.25

20% 25% 15%

20% 15% 30% 15% 13% 10% 25% 25% 35% 13% 23% 15% 17% 25% 15%

20% 5% 27% 17% 12% 20% 2.5% 8%

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Cellulose 1 and Amycoat columns 5 compounds and for Cellulose 2 and Chiralpak IC3 columns 3 compounds provided complete baseline separation with Rs  2 during the screening step. Some examples are shown in Fig. 3 for boc-saxagliptin, cinacalcet and maraviroc intermediate ester showing two different gradient conditions providing successful enantioseparations. In total, 8 compounds could be completely resolved in the screening phase using some of the 5 stationary phases, see Table 2. Among these 8 compounds 7 could be completely resolved on the two stationary phases proposed for the first-line screening. These compounds belonged among less challenging compounds based on the graphical representation in Fig. 2, except for cetirizine. It is particularly interesting to mention the cases, where the change of elution order of the two enantiomers can be observed between the two stationary phases, such as in case of cinacalcet and maraviroc intermediate ester. In enantiomeric purity control when the minor enantiomeric impurity has to be analyzed in the presence of the major enantiomer, it is more desirable to elute the minor component before the major one. This usually allows for more sensitive and reproducible quantification of the relevant enantiomeric impurity [39,40]. The separations of enantiomeric pairs with Rs < 2 required further method optimization. Fine tuning parameters, such as temperature, BPR pressure, gradient slope and change in additive concentration were considered on the stationary phases, which provided at least partial separation. The influence of the temperature was tested in the range from 10 to 60  C. Increasing the temperature reduces the mobile phase density, which results in higher retention factors. This implies that the resolution of chiral separations may improve at lower temperatures. However, the temperature effect is not always so straightforward, since it can also affect the analyte affinity for the stationary phase hereby influencing the enantioselectivity [41e43]. In agreement with some previous reports, the influence of the temperature on the enantioseparation in

SFC was very limited also in this study. As a result, in the tested set of 20 analytes the change in temperature did not improve any enantioseparation in the tested set of compounds, thus all the analyses were further performed at 40  C. Similarly, fine tuning of BPR pressure (1500e2500 psi) and change in gradient slope did not lead to any improvement of the enantioselectivity for any of tested enantiomeric pairs. The former conclusion is also in agreement with the previously published report [44], which observed the influence of the pressure on the enantioresolution to be up to 6%. It clearly indicates, that this parameter can't be successfully used as an important parameter in method optimization. Further testing included the use of partially successful gradient screening conditions (typically Amycoat or Cellulose 1 stationary phase with CO2, both tested modifiers and combined TFA/DEA additive) in the isocratic mode. However, substantially lower amount of organic modifier was applied compared to the gradient elution. This way a complete baseline separation with the Rs  2 for aprepitant intermediate, darifenacin, epinephrine, fesoterodine and its intermediate, maraviroc, tamsulosin, zolmitriptan and ambrisentan were obtained. Detailed results are shown in Table 2 and selected chromatograms in Fig. 4A. Due to the success of this approach, isocratic conditions were additionally applied also for compounds, which were successfully separated already in the screening phase to show the differences and to see the potential for further possibility to decrease the analysis time and eliminate the equilibration step in final QC method. Some examples are shown in Fig. 4B. The same compounds were selected to show the difference between gradient elution (Fig. 3) and isocratic elution. Indeed, very fast separations, sometimes between 2 and 4 min were obtained for some enantiomeric pairs, see Table 2. However, for some compounds, such as maraviroc intermediate ester, longer analysis time was needed in isocratic mode, thus the gradient method would be still the most convenient one also in the final QC method. Separations of more challenging compounds took typically 5e10 min after

Fig. 3. The chromatograms of the gradient SFC screening experiments for selected analytes e boc-saxagliptin, cinacalcet and maraviroc intermediate-ester and the structures of the analytes. Right: Amycoat column - boc-saxagliptin and cinacalcet, Chiralpak IC3 - maraviroc intermediate. Left: Cellulose 1 for boc-saxagliptin and cinacalcet, Cellulose 2 for maraviroc intermediate. The active isomers is depicted with asterisk. Detailed conditions are given in Table 2.

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Fig. 4. Chromatograms of SFC enantioseparations fine-tuned using isocratic elution: (A) maraviroc on Cellulose 1 using CO2/methanol and TFA/DEA combined additive, darifenacin on Cellulose 1 and tamsulosin on Amycoat column using CO2/isopropanol and TFA/DEA combined additive. (B) boc-saxagliptin and cinacalcet on Cellulose 1 using CO2/methanol and TFA/DEA combined additive and cinacalcet on Amycoat column using CO2/isopropanol and TFA/DEA combined additive. The active isomer is depicted with asterisk. Detailed conditions are given in Table 2.

the fine method tuning (Table 2). Another option to tune the enantioselectivity is the change in the additive concentration. It is important to note, that this

approach has an important influence only for some analytes, while in other cases the resolution is influenced only slightly as it is shown for zolmitriptan separation on Amycoat column in Fig. 5.

Fig. 5. Effect of the amount of organic modifier and the concentration of DEA on the enantioseparation of zolmitriptan on Amycoat column. (A) CO2/methanol with 0.1% TFA/DEA combined additive using 25, 20 and 15% or organic modifier. (B) CO2/methanol with increased concentration of DEA (0.5%) in the combined TFA/DEA additive using 25, 20 and 15% of organic modifier. The active isomer is depicted with asterisk.

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Fig. 6. Effect of the amount of organic modifier and the concentration of DEA on the enantioseparation of darifenacin on Amycoat column. (A) CO2/methanol and (C) CO2/isopropanol with 0.1% TFA/DEA combined additive showing the separation at 25 and 20% of organic modifier. (B) CO2/methanol and (D) CO2/isopropanol with increased concentration of DEA (0.5%) in the combined TFA/DEA additive showing the separation at 25, 20 and 35% of organic modifier, respectively. The active isomer is depicted with asterisk.

Due to basic properties of this molecule combined TFA/DEA additive in CO2/methanol at 0.1% concentration and the combined TFA/ DEA additive with increased concentration of DEA (0.5%) were compared. An increase in DEA concentration lead to substantially higher retention of both isomers and to slightly increased enantioresolution (Fig. 5). However, easier way to manipulate the enantioresolution was changing the amount of the organic modifier. Nevertheless, even slight change might sometimes be important to meet the strict requirements of regulations. Similar approach, i. e. increase in DEA concentration to 0.5% in a combined additive, was successfully applied for the analysis of indacaterol on Amycoat column (data not shown) enabling to obtain complete enantioresolution. Absolutely different behavior was observed in case of darifenacin separation on Amycoat column (Fig. 6). When increasing the concentration of DEA (0.5%) in the combined TFA/ DEA additive in CO2/methanol, the enantioresolution was completely lost (Fig. 6B), while it was around 1.6 at the concentration 0.1% of combined TFA/DEA additive (Fig. 6A). Even more interesting behavior was observed when CO2/methanol was replaced with CO2/isopropanol. The complete baseline enantioresolution with Rs  2 was obtained for both combined TFA/DEA 0.1% and 0.5% concentrations in CO2/isopropanol (Fig. 6C and D). However, the elution order of the enantiomers reversed, which was quite surprising considering quite a small concentration change. This kind of behavior was not typical and was not found to be equally effective in other cases.

The last three remaining compounds, tolterodine, ibuprofen and ketoprofen belong among the most challenging compounds in this set of analytes. Only in case of tolterodine, which was also the compound showing no separation in the screening phase, the strict requirement for Rs  2 was not met. Nevertheless, baseline separation was finally obtained when using CO2/methanol with 0.5% DEA. This option was considered in the light of the above described experiments and a broadening not yet splitting peak shape observed on Amycoat column providing the clue that the two isomers may separate. Ketoprofen and ibuprofen demonstrated partial separation when using CO2/methanol with TFA or combined TFA/ DEA additive on Amycoat column and on Chiralpak IC3. This separation seemed to be quite straightforward. However, fine tuning through isocratic elution or change in additive concentration, both using TFA and combined TFA/DEA additive, was not successful in this case. Another option to change the selectivity is the use CO2/ combined organic modifiers, such as mixtures of alcohols and acetonitrile, different alcohols or others. Both separations were finally enabled using isocratic elution with CO2/methanol:acetonitrile (1:1) and the combined TFA/DEA additive. All results are shown in Table 2. 3.3. MS compatibility of the chiral SFC screening method Coupling of separation techniques with mass spectrometry (MS) is important for the enhancement of specificity, sensitivity and

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Fig. 7. A comparison of enantioselectivity of CO2/methanol mobile phases with volatile additives ammonium formate (AmF), ammonium acetate (AmAc) and the combined TFA/ DEA additive on Cellulose 1 (A) and Amycoat (B) column. Successful separation (full enantioresolution or partial separations) is represented by the columns above the x-axis (þ), while non-successful separation is represented by the columns bellow the x-axis ().

facilitation of compound identification. While it is already quite common in achiral SFC separations, there is limited information published so far on the use of MS friendly buffer additives, such as

ammonium formate or ammonium acetate, in chiral SFC separations [45] and they have never been employed for chiral screening. Therefore, MS compatible additives ammonium acetate and

Fig. 8. Changes observed in the enantioresolution when the combined 0.1% TFA/DEA additive was replaced with volatile additives in CO2/methanol mobile phases. Separation of cinacalcet was performed on Cellulose 1 column using CO2/methanol with the addition (A) combined 0.1% TFA/DEA additive, (B) 20 mM ammonium formate and (C) 20 mM ammonium acetate. Separation of maraviroc was performed on Amycoat column using CO2/methanol with the addition (D) combined TFA/DEA additive, (E) 20 mM ammonium formate and (F) 20 mM ammonium acetate.

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Fig. 9. Effect of the injection solvent on the peak shape of atomoxetine (elution order S,R*) in gradient (A) and isocratic (B) elution using following injection solvents: heptane/ isopropanol (9:1, black, a reference in all presented chromatograms), acetonitrile (green), tetrahydrofuran (dark blue), methanol (red), ethanol (light blue) and isopropanol (violet). Injection volume was 10 ml on the Cellulose 1 column using mobile phase composed of CO2 and methanol with the combined 0.1% TFA/DEA additive in the gradient mode (5e30% in 3 min) and in the isocratic mode (20% of organic modifier). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

ammonium formate at a concentration 20 mM added to CO2/ methanol were further tested on Amycoat and Cellulose 1 stationary phases to verify the suitability of these additives for the enantioseparation. The results in Fig. 7A demonstrate that the change in enantioselectivity was very small on Cellulose 1 column. Only the enantioresolution of cinacalcet (Fig. 8) and tamsulosine was lost using whatever of the two buffers, while maraviroc intermediate ester was not separated only when using ammonium acetate. The additive comparison results were different on Amycoat column (Fig. 7B). The loss of enantioresolution was found for darifenacin, epinephrine and indacaterol using both volatile buffers. On the other hand, on this stationary phase also some enantioresolution improvements were observed when replacing the combined TFA/DEA additive in CO2/methanol mobile phase with the volatile ones. This improvement was observed for cetirizine and maraviroc (Fig. 8) with both buffers. These experiments reveal that volatile additives can also be successfully employed in chiral screening, but generally at the cost of loss of enantionselectivity for some compounds. As the results are quite encouraging and to the best of our knowledge, no reports using volatile ammonium acetate and ammonium formate in SFC chiral screening have been published so far, further research is currently being performed on this topic in our laboratory.

mode. Tested solvents included methanol, isopropanol, ethanol, acetonitrile, tetrahydrofuran and a mixture of heptane/isopropanol (9:1), which is recommended as the best solvent for SFC due to the close properties to SFC mobile phase. However, this dilution solvent is non-polar and many pharmaceutical compounds are not well soluble in such solvent. Therefore, some compromise has to be found. The choice of the solvent for the injection was found to be more critical in the isocratic mode than in the gradient elution, as it is shown in Fig. 9 for atomoxetine. As expected, alcohol solvents compromised the peak shape with the worst tendency for methanol. In the gradient elution a partial peak splitting was observed for the first eluted enantiomer, while only band broadening was observed for the second enantiomer and for both enantiomers in the isocratic elution. Tetrahydrofuran and acetonitrile were found to provide the best peak shapes in the gradient mode, where the results were comparable to the mixture of heptane/isopropanol (9:1), Fig. 9A. In isocratic elution some band broadening was observed even when using these two solvents and this phenomenon was more important for acetonitrile than for tetrahydrofuran (Fig. 9B). When tetrahydrofuran was combined with alcohols, such as methanol or ethanol, some peak broadening was still observed in both isocratic and gradient modes (data not shown). Based on these results, tetrahydrofuran was chosen as a good compromise in this study and provided overall good peak shapes for all 20 tested analytes.

3.4. Injection solvent for SFC chiral methods The importance of injection solvent in SFC have already been widely discussed in the previously published works [19,20,46,47]. For this reason a set of experiments was performed also on this topic. Several solvents typical for SFC were tested using two probe analytes, atomoxetine and cinacalcet in both isocratic and gradient

4. Conclusion Fast and efficient SFC general screening separation approach enabled by quick gradient elution was developed. The screening analysis took only 4 min followed with 2 min of column

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equilibration. Higher enantioselectivity compared to the conventional screening approaches using single acidic and basic additives was obtained when using the combined additive composed of 0.1% TFA and 0.1% DEA. Another advantage of the combined additive was no need for compound classification. This additive was systematically added to CO2/methanol or CO2/isopropanol mobile phases. Using the combination of these two mobile phases, 18 enantiomeric pairs out of 20 including all, acidic, basic and neutral structures, were at least partially resolved already in the screening phase using two stationary phases, Cellulose 1 and Amycoat. Both stationary phases contained the same 3,5-dimethylphenylcarbamoyl functional groups. The first-line screening step thus included testing of two stationary phases and two mobile phases, which fully meets the criteria of limited number of experiments needed for fast method development. If these separations were not successful, further screening should be performed using cellulose tris(3chloro-4-methylphenylcarbamate) and subsequently cellulose tris(3,5-dichlorophenylcarbamate) with the same mobile phases. While temperature, BPR pressure and gradient slope changes were found ineffective in the fine method optimization step, change in the concentration of additive, combined organic modifiers, such as methanol/acetonitrile (1:1) or use of isocratic elution at lower concentration of organic modifier were effective tools to achieve requested enantioresolution. SST results for these optimized methods demonstrated very good repeatability for both retention times and peak area. When testing volatile buffers, ammonium formate and ammonium acetate, at the place of the combined TFA/ DEA additive, quite encouraging results were obtained showing that only some enantioselectivity was lost during the screening phase. Acknowledgement The authors gratefully acknowledge research projects of Charles University in Prague UNCE 204026/2012. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.aca.2016.11.002. References [1] C. White, Integration of supercritical fluid chromatography into drug discovery as a routine support tool Part I. Fast chiral screening and purification, J. Chromatogr. A 1074 (2005) 163e173. [2] P. Franco, T. Zhang, Chapter 6: common screening approaches for efficient analytical method development in LC and SFC on columns packed with immobilized polysaccharide-derived chiral stationary phases in G. K. E. Scriba, chiral separations, Methods Mol. Biol. 970 (2013) 113e126. [3] M.B. Hicks, E.L. Regalado, F. Tan, X. Gong, Ch J. Welch, Supercritical fluid chromatography for GMP analysis in support of pharmaceutical development and manufacturing activities, J. Pharm. Biomed. Anal. 117 (2016) 316e324. [4] K.W. Phinney, SFC of drug enantiomers, Anal. Chem. 72 (2000) 204Ae211A. [5] G. Terfloth, Enantioseparations in super- and subcritical fluid chromatography, J. Chromatogr. A 906 (2001) 301e307. [6] Y. Liu, A. Berthod, C.R. Mitchell, T.L. Xiao, B. Zhang, D.W. Armstrong, Super/ subcritical fluid chromatography chiral separations with macrocyclic glycopeptide stationary phases, J. Chromatogr. A 978 (2002) 185e204. nez, Enantiomeric res[7] L. Toribio, M.J. del Nozal, J.L. Bernal, C. Alonso, J.J. Jime olution of bifonazole by supercritical fluid chromatography, J. Sep. Sci. 29 (2006) 1373e1378. [8] N. Wu, Increasing speed of enantiomeric separations using supercritical fluid chromatography, Adv. Chromatogr. 46 (2008) 213e234. [9] C. West, M. Cieslikiewicz-Bouet, K. Lewinski, I. Gillaizeau, Enantiomeric separation of original heterocyclic organophosphorus compounds in supercritical fluid chromatography, Chirality 25 (2013) 230e237. [10] A.J. Alexander, L. Zhang, T.F. Hooker, F.P. Tomasella, Comparison of supercritical fluid chromatography and reverse phase liquid chromatography for the impurity profiling of the antiretroviral drugs lamivudine/BMS-986001/ efavirenz in a combination tablet, J. Pharm. Biomed. Anal. 78e70 (2013)

209

243e251. [11] N. Matthijs, C. Perrin, M. Maftouh, D.L. Massart, Y. Vander Heyden, Definition and system implementation of strategies for method development of chiral separations in normal- or reversed-phase liquid chromatography using polysaccharide-based stationary phases, J. Chromatogr. A 1041 (2004) 119e133. [12] A. Ghanem, H.Y. Aboul-Enein, Comparison, applications, advantages, and limitations of immobilized and coated amylose tris-(3,5- dimethylphenylcarbamate) chiral stationary phases in HPLC, J. Liq. Chromatogr. Relat. Technol. 28 (2005) 2863e2874. [13] B. Chankvetadze, I. Kartozia, C. Yamamoto, Y. Okamoto, Comparative enantioseparation of selected chiral drugs on four different polysaccharide-type chiral stationary phases using polar organic mobile phases, J. Pharm. Biomed. Anal. 27 (2002) 467e478. [14] D.C. Patel, Z.S. Breitbach, M.F. Wahab, Ch L. Barhate, D.W. Armstrong, Gone in seconds: praxis, performance, and peculiarities of ultrafast chiral liquid chromatography with superficially porous particles, Anal. Chem. 87 (2015) 9137e9148. [15] Ch L. . Barhate, Z.S. Breitbach, E.C. Pinto, E.L. Regalado, Ch J. Welch, D.W. Armstrong, Ultrafast separation of fluorinated and desfluorinated pharmaceuticals using highly efficient and selective chiral selectors bonded to superficially porous particles, J. Chromatogr. A 1426 (2015) 241e247. [16] K. Lomsadze, G. Jibuti, T. Farkas, B. Chankvetadze, Comparative highperformance liquid chromatography enantioseparations on polysaccharide based chiral stationary phases prepared by coating totally porous and coreshell silica particles, J. Chromatogr. A 1234 (2012) 50e55. [17] O.H. Ismail, A. Ciogli, C. Villani, M. De Martino, M. Pierini, A. Cavazzini, D.S. Bell, F. Gasparrini, Ultra-fast high-efficiency enantioseparations by means of a teicoplanin-based chiral stationary phase made on sub-2 mm totally porous silica particles of narrow size distribution, J. Chromatogr. A 1427 (2016) 55e68. [18] A. Cavazzini, N. Marchetti, R. Guzzinati, M. Pierini, A. Ciogli, D. Kotoni, I. DAquarica, C. Villani, F. Gasparini, Enantioseparations by ultra-high performance liquid chromatography, Trends Anal. Chem. 63 (2014) 95e103. kova , A. Grand-Guillaume Perrenoud, I. Francois, C. West, E. Lesellier, [19] L. Nova D. Guillarme, Modern analytical supercritical fluid chromatography using columns packed with sub-2mm particles: a tutorial, Anal. Chim. Acta 824 (2014) 18e35. kova , Supercritical fluid [20] V. Desfontaine, D. Guillarme, E. Francotte, L. Nova chromatography in pharmaceutical analysis, J. Pharm. Biomed. Anal. 113 (2015) 56e71. [21] M. Maftouh, C. Granier-Loyaux, E. Chavana, J. Marini, A. Pradines, Y. Vander Heyden, C. Picard, Screening approach for chiral separation of pharmaceuticals. Part III. Supercritical fluid chromatography for analysis and purification in drug discovery, J. Chromatogr. A 1088 (2005) 67e81. [22] Y. Zhao, G. Woo, S. Thomas, D. Semin, P. Sandra, Rapid method development for chiral separation in drug discovery using sample pooling and supercritical fluid chromatography e mass spectrometry, J. Chromatogr. A 1003 (2003) 157e166. [23] M.S. Villeneuve, R.J. Anderegg, Analytical supercritical fluid chromatography using fully automated column and modifier selection valves for the rapid development of chiral separations, J. Chromatogr. A 826 (1998) 217e225. [24] K. De Klerck, Y. Vander Heyden, D. Mangelings, Generic chiral method development in supercritical fluid chromatography a ultra-performance supercritical fluid chromatography, J. Chromatogr. A 1363 (2014) 311e322. [25] K. De Klerck, Y. Vander Heyden, D. Mangelings, Pharmaceutcal-enantiomers resolution using immobilized polysaccharide-based chiral stationary phases in supercritical fluid chromatography, J. Chromatogr. A 1328 (2014) 86e97. [26] C.J. Welch, M. Biba, J.R. Gouker, G. Kath, P. Augustine, P. Hosek, Solving multicomponent chiral separation challenges using a new SFC tandem column screening tool, Chirality 19 (2007) 184e189. [27] Z. Pirzada, M. Personick, M. Biba, X. Gong, L. Zhou, W. Schafer, C. Roussel, C.J. Welch, Systematic evaluation of new chiral stationary phases for supercritical fluid chromatography using a standard racemate library, J. Chromatogr. A 1217 (2010) 1134e1138. [28] C. Hamman, M. Wong, M. Hayes, P. Gibbons, A high throughput approach to purifying chiral molecules using 3 mm analytical chiral stationary phases via supercritical fluid chromatography, J. Chromatogr. A 1218 (2001) 3529e3536. [29] Ch Hamman, M. Wong, I. Aliagas, D.F. Ortwine, J. Pease, D.E. Schmidt Jr., J. Victorino, The evaluation of 25 chiral stationary phases and the utilization of sub-2.0 mm coated polysaccharide stationary phases via supercritical fluid chromatography, J. Chromatogr. A 1305 (2013) 310e319. [30] Y. Zhang, W. Watts, L. Nogle, O. McConnell, Rapid method development for chiral separation in drug discovery using multi-column parallel screening and circular dichroism signal pooling, J. Chromatogr. A 1049 (2004) 75e84. [31] L. Zeng, R. Xu, D.B. Laskar, D.B. Kassel, Parallel supercritical fluid chromatography/mass spectrometry system for high-throughput enantioselective optimization and separation, J. Chromatogr. A 1169 (2007) 193e204. [32] K. De Klerck, C. Tistaert, D. Mangelings, Y. Vander Heyden, Updating a generic screening approach in sub- or supercritical fluid chromatography for the enantioresolution of pharmaceuticals, J. Supercrit. Fluids 80 (2013) 50e59. [33] K. De Klerck, D. Mangelings, D. Click, F. De Boever, Y. Vander Heyden, Combined use of isopropylamine and trifluoroacetic acid in methanol-containing mobile phases for chiral supercritical fluid chromatography, J. Chromatogr. A 1234 (2012) 72e79.

210

kova , M. Dousa / Analytica Chimica Acta 950 (2017) 199e210 L. Nova

[34] K. De Klerck, G. Parewyck, D. Mangelings, Y. Vander Heyden, Enantioselectivity of polysaccharide-based chiral stationary phases in supercritical fluid chromatography using methanol-containing carbon dioxide mobile phases, J. Chromatogr. A 1269 (2012) 336e345. [35] D. Albas, Y. Vander Heyden, M.G. Schmid, B. Chankevadze, D. Mangelings, Chiral separations of cathinone and amphetamine-derivatives: comparative study between capillary electrochromatography, supercritical fluid chromatography and three liquid chromatographic modes, J. Pharm. Biomed. Anal. 121 (2016) 232e243. [36] M.M. Wong, W.B. Holzheuger, G.K. Webster, A comparison of HPLC and SFC chiral method development screening approaches for compounds of pharmaceutical interest, Curr. Pharm. Anal. 4 (2008) 101e105. [37] D.W. Armstrong, R.M. Woods, Z.S. Breitbach, Comparison of enantiomeric separations and screening protocols for chiral primary amines by SFC and HPLC, LC-GC N. Am. 32 (2014) 742e752. [38] C. West, G. Guenegou, Y. Zhang, L. Morin-Allory, Insights into chiral recognition mechanism in supercritical fluid chromatography. II. Factors contributing to enantiomer separation on tris-(3,5-dimethylphenylcarbamte) of amylose and cellulose stationary phases, J. Chromatogr. A 1218 (2011) 2033e2057. [39] K.S. Dossou, P. Chiap, A.C. Servais, M. Fillet, J. Crommen, Development and validation of a LC method for the enantiomeric purity determination of Sropivacaine in a pharmaceutical formulation using a recently commercialized cellulose-based chiral stationary phase and polar non-aqueous mobile phase,

J. Pharm. Biomed. Anal. 54 (2011) 687e693. [40] T.J. Wozniak, R.J. Bopp, E.C. Jensen, Chiral drugs: an industrial analytical perspective, J. Pharm. Biomed. Anal. 9 (1991) 363e382. [41] P. Mourier, P. Sassiat, M. Caude, R. Rosset, Retention and selectivity in carbon dioxide supercritical fluid chromatography with various stationary phases, J. Chromatogr. A 353 (1986) 61e75. [42] U. Selditz, S. Copinga, C.J. Grol, J.P. Franke, R.A. de Zeeuw, H. Wikstrom, O. Gyllenhaal, Temperature effects on the chromatographic behaviour of racemic 2-amidotetralins on a Whelk-O 1 stationary phase in super- and subcritical fluid chromatography, Pharmazie 54 (1999) 183e191. [43] M.J. Del Nozal, L. Toribio, J.L. Bernal, C. Alonso, J.J. Jimenez, Chiral separation of omeprazole and several related benzimidazoles using supercritical fluid chromatography, J. Sep. Sci. 27 (2004) 1023e1029. [44] Ch Wang, Y. Zhang, Effects of column back pressure on supercritical fluid chromatography separations of enantiomers using binary mobile phases on 10 chiral stationary phases, J. Chromatogr. A 1281 (2013) 127e134. [45] R. Pell, W. Lindner, Potential of chiral anion-exchange operated via various subcritical fluid chromatography modes for resolution of chiral acids, J. Chromatogr. A 1245 (2012) 175e182. [46] J.N. Fairchild, J.F. Hill, P.C. Iraneta, Influence of sample solvent composition for SFC separations, LC-GC N.A. 31 (2013) 326e333. [47] V. Abrahamsson, M. Sandahl, Impact of injection solvents on supercritical fluid chromatography, J. Chromatogr. A 1306 (2013) 80e88.