# 19F-1H NMR spectroscopy in weakly orienting solvents for the enantiomeric resolution of fluorinated chiral drugs: The case of fluoxetine

Vincent Chiapolino, François-Marie Moussallieh, Philippe Lesot, Boris Gouilleux

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Vincent Chiapolino, François-Marie Moussallieh, Philippe Lesot, Boris Gouilleux. 19F-1H NMR spectroscopy in weakly orienting solvents for the enantiomeric resolution of fluorinated chiral drugs: The case of fluoxetine. Journal of Pharmaceutical Analysis, In press, 10.1016/j.jpha.2025.101469. hal-05440323

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$^{19}$F-{$^{1}$H} NMR spectroscopy in weakly orienting solvents for the enantiomeric resolution of fluorinated chiral drugs: The case of fluoxetine

Vincent Chiapolino, François-Marie Moussallieh, Philippe Lesot, Boris Gouilleux

PII: S2095-1779(25)00286-2

DOI: https://doi.org/10.1016/j.jpha.2025.101469

Reference: JPHA 101469

To appear in: *Journal of Pharmaceutical Analysis*

Received Date: 15 March 2025

Revised Date: 3 October 2025

Accepted Date: 9 October 2025

Please cite this article as: V. Chiapolino, F.-M. Moussallieh, P. Lesot, B. Gouilleux, $^{19}$F-{$^{1}$H} NMR spectroscopy in weakly orienting solvents for the enantiomeric resolution of fluorinated chiral drugs: The case of fluoxetine, *Journal of Pharmaceutical Analysis*, https://doi.org/10.1016/j.jpha.2025.101469.

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© 2025 Published by Elsevier B.V. on behalf of Xi'an Jiaotong University.
¹⁹F-{$^{1}$H} NMR spectroscopy in weakly orienting solvents for the enantiomeric resolution of fluorinated chiral drugs: The case of fluoxetine

Vincent Chiapolino<sup>a</sup>, François-Marie Moussallieh<sup>a</sup>, Philippe Lesot<sup>a, b, **</sup> and Boris Gouilleux<sup>a, *</sup>

<footnote><sup>a</sup> University of Paris-Saclay, Institute of Molecular Chemistry and Materials of Orsay (ICMMO), Orsay, 91400, France</footnote>

<footnote><sup>b</sup> National Center for Scientific Research (CNRS), Paris, 75016, France</footnote>

<footnote>* Corresponding author.</footnote>

<footnote>** Corresponding author.</footnote>

<footnote>University of Paris-Saclay, Institute of Molecular Chemistry and Materials of Orsay (ICMMO), Orsay, 91400, France.</footnote>

<footnote>E-mail addresses: boris.gouilleux@universite-paris-saclay.fr (B. Gouilleux),
philippe.lesot@universite-paris-saclay.fr (P. Lesot).</footnote>
<figure><img src="image_3.png" /><figcaption>Chiral API dissolved in a
lyotropic liquid crystal (PBLG/CHCl₃)</figcaption></figure>

¹⁹F-{*H} NMR experiment

Enantiomeric discrimination
with high resolution

FT and
processing

¹⁹F-{*H} NMR
spectrum

δ³F (ppm)

<figure><img src="image_4.png" /></figure>
# ¹⁹F-{¹H} NMR spectroscopy in weakly orienting solvents for the enantiomeric resolution of fluorinated chiral drugs: The case of fluoxetine

## Abstract

A rapid and simple method combining ¹⁹F-{¹H} nuclear magnetic resonance (NMR) and weakly orienting chiral solvents is proposed for the spectral discrimination and accurate quantitation of fluoxetine enantiomers. Fluoxetine (FLX) is a well-known bioactive chiral drug in pharmacology with well-established anti-depressant properties (Prozac or Sarafem) and currently used as a racemate. Since 1991, it has been established that the (S)-form of FLX shows different pharmacokinetic and pharmacodynamic profiles in comparison to the (R)-form, hence the development of novel enantioresolved NMR methods for analyzing FLX is of interest. The reported approach, relying on a commercial polymer: poly-γ-benzyl-L-glutamate (PBLG) as chiral selector, addresses the enantiomeric analysis of FLX hydrochloride solutions with a high level of accuracy in a short time. The influence of experimental parameters, such as solute concentration and temperature on the enantiomeric resolution is investigated and discussed in depth. In particular, the uniformity and stability of PBLG-based lyotropic liquid crystals (LLCs) in presence of a hydrochloride analyte is assessed for the first time by ¹⁹F NMR imaging. All the outcomes obtained highlight the analytical potential of this NMR approach for the enantiomeric analysis of fluorinated chiral drugs while the spectral data recorded at various conditions in temperature and mesophase composition provide new valuable insights toward a better understanding of chiral recognition processes in polypeptide orienting media.

**Keywords:** Fluorinated chiral drugs, Fluoxetine, ¹⁹F NMR, PBLG-based LLC, Enantiomeric analysis, Active pharmaceutical ingredient

## 1. Introduction

Molecular chirality plays a pivotal role in pharmaceutical chemistry, with over 50% of the drugs currently on the market containing at least one stereogenic center [1,2]. While one enantiomer of a chiral active pharmaceutical ingredient (API) leads to the desired activity on the biological target (eutomer), the other may induce disparate effects, ranging from no activity to opposite or even toxic side-effects (distomer) [3]. Consequently, an increasing number of innovative chiral drugs are being marketed as pure enantiomeric compounds, while some drugs initially used as a racemate are being replaced by the identified eutomer (i.e., chiral switch) to improve the therapeutic efficacy [4,5]. This involves the continued development of rapid, versatile and complementary analytical tools to determine the
enantiomeric purity of a bioactive substance at various stages, from drug synthesis steps to the control of the final commercial medicines [6].

The main analytical methods currently available for the chiral analysis include chiroptical spectroscopy, chiral gas or liquid chromatographies, chiral capillary electrophoresis, chiral mass spectrometry or liquid-state nuclear magnetic resonance (NMR) in presence of a chiral agent (CA) [7,8]. These CAs can be, on the one hand, chiral derivatization agents (CDAs) such as Mosher acid leading to diastereoisomers by covalent bonding and [9], on the other hand, chiral metal complexes, lanthanide shift reagents, chiral solvating agents (CSAs) known to form diastereoisomeric adducts by means of intermolecular interactions [10–15]. The conversion of enantiomers into diastereomeric entities allows generally the NMR spectral discrimination of enantiomers by variations in their isotropic chemical shifts or those of the CA. Most of the developed CAs offer effective enantiomeric discrimination for only one class of compounds, such as carboxylic acids [16,17], amines [13,18–21], aliphatic alcohols [9], although cyclodextrins [22] or the recently reported benzamide-based CSAs [23] may be suitable for a wider range of chiral analytes. In addition, a significant number of CSAs reported in the literature have been specifically synthesized and are not commercially available at this time [24]. For them, the users must therefore carry out their synthesis and purification prior to be exploitable in chiral analysis, hampering somewhat the application of this methodology in routine.

Interestingly, the concept of enantiospecific non-covalent interactions between stereoisomers and a CA in isotropic solvents can be extended to orienting media such as chiral lyotropic liquid-crystalline phases based on an enantiopure helical polymer (i.e., the chiral selector) dissolved in routine organic solvents (polar or weakly polar) in appropriate proportions [25,26], or based on smaller non-polymeric entities undergoing a self-assembling process [27,28]. In such chiral weakly orienting media, the chiral analyte adopts an average orientational order that varies from one enantiomer to another [29–32]. This divergence in the molecular orientation results in a difference of three major residual anisotropic NMR interactions, namely residual chemical shift anisotropy (RCSA), residual dipolar coupling (RDC) and residual quadrupolar coupling (RQC), which is at the base of the enantiomeric discrimination in chiral anisotropic media [25,26].

In the frame of the enantiomeric analysis of chiral fluorinated APIs ($^{19}$F-APIs), we pioneered in 2024 a promising approach involving anisotropic $^{19}$F one-dimensional (1D) NMR and a chiral lyotropic liquid crystal (LLC) based on poly-γ-benzyl-L-glutamate (PBLG) [33]. The molecular structure of the PBLG is shown in Fig. 1A. First experimental results obtained with Flurbiprofen and Efavirenz demonstrated the applicability of the anisotropic NMR approach to chiral APIs involving diverse chemical features, such as
carboxylic acid, cyclic carbamate, aromatic and acetylenic groups. This versatility is also extended to the case of alcohols as presented here with the enantiomeric resolution of 2,2,2-trifluoro-1-phenylethan-1-ol (TFPE) shown in Fig. 1B.

To further highlight the relevance of this methodology in the frame of chiral APIs, we have explored the case of fluoxetine (FLX) (Fig. 1C), a selective serotonin reuptake inhibitor mainly used for the treatment of major depression and obsessive-compulsive disorder [34,35]. The (S)-enantiomer of FLX has a longer duration of action and the (S)-form of the active metabolite (norfluoxetine) has a superior potency as serotonin inhibitor than its (R)-counterpart [36,37]. In addition to the importance of proposing alternative methods for the chiral analysis of this widespread drug, FLX offers two interesting chemical features that may challenge the herein approach. First, this compound includes an amine function which can cause detrimental line-broadening on the NMR spectrum when dealing with polypeptide-based LLCs prepared with apolar solvents (i.e., chloroform, dichloromethane) as previously reported [38]. Second, FLX is usually marketed in a hydrochloride form to achieve desirable formulation properties, such as a greater solubility in water. Dissolution of hydrochloride salts in polypeptide-based LLCs has been very rarely described to date, and hence the potential impact of HCl salts on the stability and uniformity of the liquid-crystalline phase deserves to be investigated.

In this article, we present an exhaustive study on the spectral discrimination properties and quantitation of FLX enantiomers by the combination of ¹⁹F-{¹H} 1D NMR and a PBLG/chloroform-based LLC, examining various key experimental parameters (temperature and solute amount). To illustrate the analytical value of this approach, all ¹⁹F spectral data under discussion have been obtained: i) by the means of routine 1D ¹⁹F NMR experiments within short acquisition times of no more than a few minutes; ii) after a relatively simple sample preparation involving a commercially available polymer without the need of further purification and with the possibility to use non-deuterated solvents; and iii) by using only a routine medium-field NMR instrument (300 MHz spectrometer, i.e., 7.05 T, and room-temperature classical NMR probe), demonstrating the suitability of this approach on an NMR equipment representative of common (academic or pharmaceutical) analytical platforms. The ability of yielding uniform and stable mesophases in presence of hydrochloride and amine-contained molecules will be also investigated by ¹⁹F NMR imaging. The complex role of the solute concentration and temperature onto the resulting enantiomeric resolution is explored in details to provide the best experimental conditions. Finally, the quantitative performance of the enantiopurity determination of APIs using this enantioresolved method will be evaluated in terms of trueness and precision.

## 2. Material and methods
## 2.1 Chemicals

(Rac)-FLX and (S)-FLX were purchased at Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan) and at Biosynth (Staad, Switzerland), respectively, both with a purity of 98%. TFPE was purchased at Acros Organics (Geel, Belgium). The PBLG polymer was synthesized by the synthesis platform of the Institute of Molecular Chemistry and Materials of Orsay (ICMMO). The degree of polymerization (DP) of the PBLG used in this study was estimated at 641 by ¹H diffusion ordered spectroscopy (¹H DOSY) NMR. Note here that PBLG is also commercially available (for instance at Merck or Sigma-Aldrich). Chloroform (Carlo Erba, Cornaredo, Italy) used to prepare the liquid-crystalline phases was stabilized with ethanol (<0.004%) and dried on molecular sieves. All compounds listed above were used without any further purification.

## 2.2 Sample preparation

The oriented samples were prepared by dissolving an amount of FLX (< 30 mg) into dry CHCl₃ in a flat vial, then the solution was added into a 5-mm NMR tube containing nearby 100 mg of PBLG. The exact amount of chloroform (about 600 mg) was adjusted so that the total weight% of polymer (i.e., mass of PBLG/total mass of sample components) was maintained at 14%, leading to a sample length of approximately 3.5 cm. The exact compositions of each sample are given in Tables S1 and S2. The NMR tube was fire-sealed to avoid CHCl₃ evaporation over time and then several low-speed centrifugation cycles (e.g., 500 rpm during 30 s) with inversion of the sample between each cycle were carried out to remove matter gradients (Fig. S1). The centrifugation protocol explored in this work and its impact on the liquid crystal uniformity is discussed in the Section 3 (see also in the Supplementary data). Once prepared, the samples were stored in the absence of light and at room temperature until analysis.

## 2.3 ¹⁹F NMR experiments

All ¹⁹F NMR experiments were conducted on a 7.05 T ($ν_0$ (¹⁹F) = 282.4 MHz) Bruker NEO spectrometer equipped with a 5-mm BBFO probe (Bruker Corporation Company, Billerica, Massachusetts, U.S.). ¹⁹F-{*}H{¹H} 1D NMR spectra were recorded with a 90° radiofrequency pulse length of 15 µs (power of 6.9 W) and the free induction decay (FID) was sampled during 1 s with a ¹H composite pulse decoupling (WALTZ-16) (the zgig sequence in the Bruker's Topspin library). The inter-scan delay was shorter than the usual 5 × T₁ required in quantitative NMR. This improves the signal-to-noise-ratio (SNR) *per* time unit without inducing a quantitative bias due to similar fluorine longitudinal relaxation, T₁(¹⁹F) for an enantiomeric pair (see Section 3).
Given their short experimental duration (e.g., a few minutes), ¹⁹F-{¹H} spectra are not impacted by the natural drift of the magnetic field $B_0$, and hence all experiments can be carried out without deuterium lock. The samples can be therefore prepared without deuterated chloroform and without generating any undesired solvent signals on the ¹⁹F NMR spectra. Under this condition, the shimming process is achieved via an automatic shimming procedure applied on the ¹H signal of $CHCl_3$ to optimize the signal line-shape. Note that the achievement of this last step highly depends on the spatial uniformity of the liquid-crystalline phase [39]. The FIDs were processed using the Mnova software 14.2 (Mestrelab, Santiago de Compostela, Spain), including zero-filling (128 k datapoints), Fourier Transform, manual phasing and a polynomial (order 5) baseline correction.

The signal integration for the measurement of enantiomeric excess (ee%) was achieved by a line-fitting procedure to minimize area biases in cases of enantiomeric signals non-fully baseline separated. This data-processing fits the NMR peaks to a linear combination of Lorentzian and Gaussian functions and finely tunes the height, the linewidth and the Lorentzian/Gaussian ratio. The linewidth and Lorentzian/Gaussian ratio were not imposed constant for all components of a same ¹⁹F triplet in order to take account of any asymmetry (even slight) due to imperfect mesophase uniformity or differential line broadening effects (i.e., cross-correlated relaxation). The $T_1(^{19}\text{F})$ values of FLX enantiomers were measured by standard inversion-recovery experiments carried out with 16 incremented inversion delays. 16 scans per increment were accumulated with a repetition time of 10.5 s ensuring a full longitudinal relaxation between scans. The pseudo-2D data were processed by the $T_1/T_2$ dynamic module of Topspin 4.0 (Bruker) where a standard mono-exponential function was used for non-linear fitting.

The Z-imaging ¹⁹F NMR experiments were carried out by 17 successive slice-selective experiments along z-axis (i.e., along the NMR tube length) with a thickness of 0.1 cm, each. These slice selections are obtained by a concurrent application of a pulse-field-gradient (22.7 G/cm) and a shaped-pulse (“qsneeze”) of 0.8 ms inducing an excitation bandwidth of 10 kHz. The slice position from the bottom to the top of the sensitive volume of the NMR probe was incremented via the shaped-pulse offset frequency ranging from -80 to 80 kHz.

# 3. Results and discussion

## 3.1 Enantiomeric discrimination by anisotropic ¹⁹F NMR observables

Within an achiral isotropic medium, the ¹H-decoupled ¹⁹F NMR spectrum of molecules containing a $CF_3$ group is reduced to a single resonance centered on the isotropic ¹⁹F chemical shift, $\delta_{iso}(^{19}\text{F})$ associated to three homotopic fluorine nuclei ($A_3$ spin system). The same mixture dissolved in the PBLG/CHCl₃ phase leads to a more complex spectral pattern
where the CF₃ group leads to a (1:2:1) triplet centered on the anisotropic ¹⁹F chemical shift, δaniso(¹⁹F). The successive lines of triplet are separated by the ¹⁹F–¹⁹F total spin-spin coupling constant, TFF (observed in absolute value), which is equal to three times the residual ¹⁹F–¹⁹F dipolar coupling, DFF, between the equivalent fluorine nuclei:

$$T_{FF}(R \text{ or } S) = 3D_{FF}(R \text{ or } S) \qquad \text{Eq. (1)}$$

When chiral recognition occurs between enantiomers, a pair of non-superimposable triplets centered on δaniso(S) and δaniso(R) are expected, one corresponding to one enantiomer. Spectral enantiomeric discrimination is therefore achieved through differences of both residual ¹⁹F chemical shift anisotropy ¹⁹F-RCSA and residual ¹⁹F–¹⁹F dipolar coupling (¹⁹F–¹⁹F)-RDC. This concept is well illustrated in the case of TFPE (Fig. 2A) and FLX (Fig. 2B), two compounds involving various functional groups. The (R)- and (S)- enantiomers assignment can be achieved by comparison with scalemic series involving a small ee% (approximately 10 %), such as in the case of FLX (Fig. 2B). In this ¹⁹F–{¹H} spectrum, recorded at 300 K with a lyotropic liquid-crystalline phase containing 3.3 mg (i.e., 23.8 mmol/L) of FLX dissolved in PBLG/CHCl₃ with a 14% mass ratio in polymer, the variations ΔΔδ(R, S) and |TFF(R)| – |TFF(S)| are equal to 0.09 ppm and 34.1 Hz, respectively. Given the spectrometer magnetic field (B₀ = 7.4 T, ν₀(¹⁹F) = 282.4 MHz), ΔΔδ(R, S) corresponds here to 25.4 Hz, so that the (¹⁹F–¹⁹F)-RDC is here the key NMR observable in the spectral enantiomeric discrimination of FLX.

The underlying mechanisms governing chiral recognition in polymer-based LLCs are not yet fully understood, and deciphering them remains an on-going challenge, as evidenced by recent theoretical work based on molecular dynamics simulations [40]. We have explored the FLX-PBLG interactions to get some insights on the spatial arrangement of this API at the vicinity of PBLG side chains and to track potential discrepancies between enantiomers explaining the chiral discrimination. This study, based on saturation transfer difference (STD) NMR experiments [41–43], is presented in detail in Supplementary data. In brief, we can outline an arrangement of FLX in contact with PBLG, in which aromatic groups of FLX interact with the terminal phenyl of PBLG while the amino alkyl chain of the API is located close to the carbonyl part of the polymer (Fig. S2). The asymmetric center of FLX is at similar distance from the ester and aromatic groups of PBLG. The same trend is observed for both enantiomers with only slight differences, suggesting a similar arrangement of FLX enantiomers in contact with the PBLG side chains. In spite of these minor variations, the enantiomeric resolution of FLX is well observed as seen in Fig. 2B.

Returning to the anisotropic FLX spectrum, it is worth noting the significant discrepancies in the relative peak intensities observed between the central and external
components of triplets compared to the expected (1:2:1) ratio. The line-widths measured at
half maximum are clearly different for the deshielded, central and the shielded components
of the triplet, revealing a differential line-broadening within a same triplet. This feature is
not related to an inappropriate magnet shimming process or partially inhomogeneous
samples as this feature remains regardless the spectrometer used and the operator involved.
This differential line-broadening is complex and probably arises from interferences of
relaxation processes that can occur in case of nuclei involving a strong shielding anisotropy,
such as ¹⁹F, ³¹P, or ¹⁵N [44]. Previous observations of differential line-broadening have been
also reported in liquid-state NMR considering fluorinated-aromatic compounds with
various spin-systems (e.g., AMX and AX₂) due to cross-correlation between dipole-dipole
and dipole-CSA relaxation mechanisms [45,46].

To investigate the potential cross-correlation relaxation in the present case, inversion-
recovery experiments have been carried out to extract the longitudinal relaxation times
T₁(¹⁹F) for each component of a given triplet. The values measured vary from one line to
another within the same triplet that is consistent with cross-correlated relaxation (Table 1).
In the herein chiral environment, this cross-correlation effect is similar for the both
enantiomers, so it cannot be used for (R)- and (S)-stereoisomers assignment. In the same
vein, the T₁(R) and T₁(S) show no differences (or at least within the uncertainty of the
measurement) in longitudinal relaxation of the enantiomers (Table 1 and Fig. S3).

3.2 Spatial uniformity and stability of LLCs

LLCs based on polypeptides such as the PBLG polymer with a positive molecular
magnetic susceptibility anisotropy (Δχ<sub>m</sub> >0) lead to cholesteric mesophases that are
transformed almost instantaneously into chiral nematic media with director, ⃗n, parallel to
B<sub>0</sub> under the effect of an intense magnetic field [47]. The spatial uniformity of such
mesophases is of primary importance on the quality of the resulting NMR spectra. As
studied by Gil and co-workers [39], inhomogeneities cause a variation of the orientational
ordering of the guest molecules (solute and co-solvent) according to their spatial position in
the sample. In such case, the residual anisotropic NMR interactions become spatially
dependent leading to severe line-broadening and strong signal distortions of resonances
along with possible complications for the shimming process.

The characteristics (uniformity and stability) of the PBLG liquid-crystalline phases in
presence of HCl salts as solute are not well-known. A previous report from Samulski and
co-workers [48] underlined the significant impact of trifluoroacetic acid on the Δχ<sub>m</sub> of
PBLG and also on the mesophase fluidity. Thiele and co-workers [49] further explored the
influence of various acids and showed the destabilization of PBLG-based liquid crystals by
helix-to-random-coil transition with increasing acid content or the acid strength (example of TFA). In this context, to ascertain the quality of the prepared PBLG-based LLCs in presence of hydrochloride salt of FLX, one-dimensional Z-imaging ¹⁹F NMR has been applied at different days since sample preparation in order to characterize the uniformity and stability of the mesophases. This fluorine-19 Z-imaging 1D experiment consists in recording a series of ¹⁹F-{¹H} spectra slice by slice along Z-axis (i.e., along the NMR tube) (Fig. S4). The resulting data are presented as pseudo-2D spectra (δ¹⁹F, Z) in contour 2D plot with horizontally the ¹⁹F NMR dimension and vertically the position along Z-axis.

Immediately after the sample preparation (sealed tube) and without any homogenization procedure, the resulting mesophase is severely inhomogeneous (matter gradients) and the ¹⁹F-{¹H} spectra recorded on the whole sensitive volume of the NMR probe are not exploitable (Fig. 3A), even after several days (Fig. S5). Multiple cycles of centrifugation at moderate rotation speed (e.g., 10 times 30 s at 500 rpm) with inversion of the sample between each cycle, generally leads to a spatially uniform mesophase. This is shown in (Fig. 3B) where parallel straight lines along Z-axis and an overall ¹⁹F-{¹H} spectrum with suitable line-shape and resolution are obtained. Alternatively, when centrifugation of the NMR tube is not possible (no centrifuge available), manual homogenization based on gravity flow of the mesophase can be envisaged, especially in cases of fluid samples. For FLX samples, flipping the NMR tube up and down several times (e.g., 10 times) before NMR experiments leads also to a uniform mesophase as soon as three days after the sample preparation with comparable results (Fig. S6). Note that at the day of the sample preparation, such a manual homogenization procedure does not produce a sufficient uniformity for a suitable enantiomeric resolution (Fig. 3C). This issue could be, however, overcome by targeting only a uniform portion of the sample using spatially-resolved NMR experiments [50]. This strategy is currently in progress.

Regarding the stability, ¹⁹F NMR and Z-imaging experiments have been performed until ten days after the sample preparation. No degradation of the mesophase has been observed in this time-scale while the spatial uniformity has been maintained (Fig. S7). To check whether this uniformity is maintained under varying experimental conditions, a second LLC sample with a different FLX concentration (36.1 mmol/L) has been monitored at two different temperatures (27 and 37 °C). The ¹⁹F Z-images clearly show (Fig. S8) uniform LLCs at this concentration and for both temperatures.

In overall, these results demonstrate that the presence of FLX as an HCl salt does not prevent the production of uniform liquid crystals stable over time, which are robust again changes in experimental conditions.
### 3.3 Optimization of the enantiomeric resolution

To improve the reliability of *ee%* determination in the case of FLX, the enantiomeric resolution of anisotropic ¹⁹F-{¹H} experiments is mainly optimized as a function of the concentration in solute and sample temperature. These two experimental parameters have recently shown significant effects on the average orientational order adopted by some chiral APIs in a PBLG-based mesophase [33]. Another parameter impacting the magnitude of RCSA and RDC is the concentration in polymer into the organic solution containing a helicogenic solvent (here CHCl₃) and the analyte. The higher the concentration, the greater the degree of orientation of the mesophase (and so the magnitude of anisotropic NMR observables of analytes) [51], but at the expense of an undesired line-broadening due to higher viscosity of the medium. A good balance lies in a mass ratio of 12%-15% in PBLG, leading to a stable aligning system (without isotropic domain) while reasonable linewidths varying from 1.5-5 Hz at half-maximum are observed. In what follows, all PBLG samples have been prepared with a constant mass ratio of polymer of 14%. The sample compositions are detailed in Tables S1 and S2.

### 3.4 Effect of solute concentration

The effect of concentration of analyte into the PBLG-based LLCs has been examined with an increasing amount of FLX from 1 mg (i.e., 8.0 mmol/L) to 30 mg (i.e., 216.3 mmol/L). As highlighted in Fig. 4A, the solute concentration has here an impact on the measured $|T_{FF}|$ according to the concentration range. Slight variations are observed at high concentrations of FLX while the $|T_{FF}|$ values significantly drop for a FLX content lower than 10 mg (i.e., 72.2 mmol/L). It must be noted that these variations of residual ¹⁹F-¹⁹F dipolar coupling are different from one enantiomer to another (Fig. 4B). It is therefore possible to enhance the enantiomeric resolution by tuning the FLX concentration in the mesophase. This variation of $|T_{FF}|$ between enantiomers is more accentuated for samples at lower FLX concentrations. For instance, in the range of 1-10 mg of FLX (i.e., 8.0-72.2 mmol/L), large differences of total coupling constant between enantiomers ($|T_{FF}(R)| - |T_{FF}(S)|$) are observed, up to a 53 Hz difference. As the dipolar coupling interaction does not depend on the strength of the magnetic field $B_0$, such a frequency difference could be suitable for the enantiomeric analysis with spectrometers operating at lower magnetic fields.

The difference of anisotropic chemical shift between the two enantiomers $|\Delta\Delta\delta(R, S)|$ evolves in a similar manner with respect to the FLX concentration. Slight variations are observed at high concentrations: from $|\Delta\Delta\delta(R, S)| = 0.02$ ppm at 30 mg (i.e., 216.3 mmol/L) to 0.04 ppm at 10 mg (i.e., 72.2 mmol/L) while the anisotropic chemical shift differences between enantiomers increase significantly at lower FLX contents reaching
$|\Delta\Delta\delta(R, S)| = 0.14$ ppm for 1 mg of FLX (i.e., 8.0 mmol/L). All the spectral data in terms of coupling values and difference of chemical shifts are listed in Tables S3–10.

Based on these observations, the concentration range 8.0–72.2 mmol/L leads to a high enantiomeric resolution while the sensitivity remains sufficiently high for quantitative analyses, even with a short experimental duration. The variation of these anisotropic observables with respect to the solute concentration relies on a complex interplay between solute-solute, solute-solvent and solute-polymer interactions, which is rather difficult to decipher and simply rationale to date. However, it is possible to explore certain hypotheses in an attempt to interpret these results. The first point to be investigated is the impact of the solute on the orienting properties of the liquid-crystalline phase. As previously established, the presence of small quantities of acid (e.g., 2–10 %weight ) can significantly disturb the orienting properties of PBLG mesophase (in particular the degree of order) manifested by a drop of the residual $^2$H quadrupolar coupling ($^2$H-RQC) observed on the $^2$H signal of CDCl$_3$ when used as organic co-solvent [49]. As an hydrochloride salt, FLX may have an impact to the mesophase orienting properties, especially at high content such as 4 %weight (i.e., 30 mg of FLX). To address this question, the $^1$H–$^{13}$C spin-spin total coupling of the co-solvent (here CHCl$_3$) defined as $T_{CH} = J_{CH} + 2D_{CH}$, directly measured on the $^1$H NMR spectra (signal of $^{13}$C-satellites of the solvent), has been tracked as a function of the FLX concentration (Fig. S9). As seen, the absolute values of this anisotropic observable remain quite constant on the FLX-concentration range explored with a relative standard deviation (RSD) of 0.76% (Fig. S10 and Table S11), thus excluding a significant modification of the mesophase orienting properties through FLX adding.

The variation of the anisotropic observables of FLX with respect to its concentration is thus intrinsic to the solute, and hence the decrease in $D_{FF}$ should be discussed from the analysis of the Eq. 2:

$$D_{FF} = -k_{FF} \times \frac{S_{FF}}{r_{FF}^3} \quad \text{with} \quad S_{FF} = \left\langle \frac{3\cos^2\theta_{FF}^{B_0}-1}{2} \right\rangle \qquad \text{Eq. (2)}$$

In this equation, $r_{FF}$ is the distance between each fluorine atom within the CF$_3$ group, $S_{FF}$ is the local order parameter associated with the internuclear direction between the coupled fluorines, $\theta_{FF}^{B_0}$ is the angle between this internuclear direction and the magnetic field B$_0$ axis, and $k_{FF}$ is a constant related to the dipolar interaction ($k_{FF}$= 106.30 kHz.Å$^3$) (Figs. 5A and B). The brackets $<...>$ stand for an ensemble average.

Analysis of Eq. 2 indicates that the reduction of the RDC can be due to either a reduction of the generalized degree of order (GDO) experienced by the analyte in average,
or a progressive change of the orientation of this molecule as the $\theta_{FF}^{B_0}$ angle approaches the magic angle $\theta_m$, which in turn cancels the dipolar interaction (Fig. 5C). Under the latter hypothesis, after decreasing continuously to zero, the ($^{19}$F−$^{19}$F)-RDC should then increase (in absolute value) as $\theta_{FF}^{B_0}$ would move away from $\theta_m$. This investigation has been thus pursued with further diluted samples (i.e., lower than 8.0 mmol/L). The $^{19}$F−{$^1$H} NMR spectra obtained are markedly different from those obtained in the previous concentration range of 8.0–216.3 mmol/L (Fig. 4A). If we now examine the changes in the dipolar coupling over this wide range of concentrations, focusing for example on the (S)-isomer, we can observe that $|T_{FF}(S)|$ decreases continuously up to a certain concentration ($\theta_{FF}^{B_0}$ approaching $\theta_m$), then increases (in absolute value) at lower concentrations ($\theta_{FF}^{B_0}$ moves away from $\theta_m$). This observation is therefore consistent with a progressive reorientation of this enantiomer with respect to the FLX concentration. Note that this variation would involve a sign change of the $T_{FF}$ value that is not directly visible on the $^{19}$F−{$^1$H} NMR spectra as the coupling constants are measured in absolute value. Yet, a sign change is here consistent with the opposite differential line-broadening observed in the spectrum at the bottom of Fig. 4A compared to those shown at higher concentrations, since the line-broadening/narrowing of the shielded/deshielded components of a signal induced by cross-correlation relaxation depends on the sign of the coupling [44].

### 3.5 Temperature effect

Another key parameter controlling the orientation properties of the LLC and, subsequently the analyte, is the sample temperature. A new investigation ascertains the influence of temperature (T) on the same anisotropic observables has therefore been conducted. In principle, both RCSA and RDC can be used with that respect. However, the anisotropic chemical shift observed in LLCs implies an isotropic and an anisotropic contribution of the shielding tensor, both of which can vary with temperature. This complicates therefore the analysis of the temperature effect on the solute orientation via changes in anisotropic chemical shift. In contrast, dipolar coupling does not contain any isotropic character, making the $T_{FF}$ coupling constant a much better probe for studying solute orientation under temperature changes. At high concentrations (72.2 to 216.3 mmol/L), the coupling values $|T_{FF}|$ measured for each enantiomer decrease slightly linearly with T (Figs. 6A and B). This trend is intuitively expected, since increasing temperature leads to a reduction in the degree of order of the liquid-crystalline phase until a state of isotropy is reached. Since the slope of the linear function for each enantiomer, around 0.5 Hz/°C, is similar (blue and red curves in Fig. 6B), the difference in dipolar coupling between enantiomers is considered as constant over this temperature range of 24–42 °C. This behavior is observed for all FLX concentrations, from 216.3 to 23.8 mmol/L.
Interestingly, in the case of diluted samples (e.g., 0.4 mg of FLX), the effect of *T* is rather unexpected since the variation of |*T*<sub>FF</sub>| of the CF<sub>3</sub> group is now enantiospecific (Figs. 6C and D). Indeed, the slope of the |*T*<sub>FF</sub>| linear variation with *T* is now significantly different (−1.4 vs. 2.9 Hz/°C) from one enantiomer to another (see red and blue plots in Fig. 6D). This implies that the temperature can be adjusted to improve the enantiomeric resolution at concentrations of FLX lower than 8.0 mmol/L. More fundamentally, this unexpected increase of |*T*<sub>FF</sub>| experienced by the (S)-isomer with temperature could be explained by a change of its average orientation in the mesophase influencing the local order parameter ⟨*S*<sub>FF</sub>⟩ associated with its CF<sub>3</sub> group. This phenomenon apparently predominates over the reduction of the global decrease in ordering in this restricted temperature range (24–42 °C). An increase in *T* well above 42 °C would ultimately lead to a decrease in the values of |*T*<sub>FF</sub>| for both enantiomers as we approach the transition temperature to the isotropic state.

3.6 Quantitation of enantiomers in racemic and scalemic series

The determination of the *ee%* of an API by NMR is by essence a quantitative
measurement. NMR spectroscopy is a well-established quantitative technique, provided
certain conditions are respected during acquisition and processing step. Mastering the
potential quantitative biases deserves therefore attention in the sake of accurate
measurement of the *ee%*. The most important one is the uncompleted longitudinal relaxation
during the repetition time (*T*<sub>R</sub>) between two successive scans, at various extent between
nuclei with different characteristic relaxation times *T*<sub>1</sub>. In quantitative NMR experiments,
this bias is usually circumvented with a long repetition time *T*<sub>R</sub> fixed at five times the longest
*T*<sub>1</sub> of nuclei to be probed. However, this quantitative condition is yielded at the expense of
SNR per time unit.

This annoying but necessary requirement can be alleviated in the case of a relative
quantification of enantiomers because no significant differences of relaxation times T₁
between enantiomers have been so far reported (also in weakly aligning LLCs), at the best
of our knowledge. This is again verified here in the case of FLX where no differences of
T₁(<sup>19</sup>F) (greater than the measurement uncertainty) have been observed between the two
enantiomers. This occurrence allows to record <sup>19</sup>F-{<sup>1</sup>H} 1D NMR spectra in partial
saturation conditions without inducing a systematic quantitative bias. A significant
improvement in the SNR *per* time unit is therefore gained, thus enhancing the repeatability
(precision) and detection limit of the method for a given experimental duration, *t*exp.
<sup>19</sup>F-{<sup>1</sup>H} 1D NMR experiments (*t*exp. ≈ 4 min) were performed on both racemic and
scalemic series and the experimental *ee%* determined as follows:
$$ ee(i)\% = 100 \times \frac{A_i - A_j}{A_i + A_j} \qquad \text{Eq. (3)} $$

where the subscript “i” is related to the enantiomer in excess (major isomer) and A is the signal area. In practice, the signal integration is performed by a line-fitting procedure (Fig. 7) where the experimental lines are reconstructed by a linear combination of Lorentzian and Gaussian functions.

The trueness of the method, i.e., deviation between the expected and experimental values of the measurement, was evaluated only on the racemic sample as the expected ee = 0% does not suffer from weighting uncertainties during the sample preparation. In practice, the deshielded components of the two triplets were selected for this purpose because they provide the best enantiomeric resolution (but not the better SNR). The precision (here considered as the short-term repeatability) is determined by the standard deviation (SD%) of the measurement calculated on a series of five successive identical NMR experiments.

The trueness determined on the racemic mixture is of 0.09% (Table 2, entry 1), which highlights the excellent agreement between theoretical and experimental ee% values and the absence of quantitative biases. This high level of accuracy is achieved without any prior calibration procedure, reference materials or post-correction factors.

For an experimental duration of 4 min at 282.4 MHz, the SNR obtained on the signals of FLX enantiomers is of the order of 1800 (for the central lines of the triplets) and about 950 for the deshielded components at a concentration of 36 mmol/L per enantiomer (see entry 1 of Table 2). This ensures an excellent precision (SD = 0.08%) for this 10 mg FLX sample, which enables here the measurement with an uncertainty of ±0.16%, if a 95% interval confidence is considered (calculated as 2 × SD (%)). Interestingly, the SD (%) value does not vary significantly with the total mass of FLX for scaleic samples, as seen in Table 2, by comparing entry 2 (15 mg of FLX) and entry 3 (10 mg of FLX). Despite a difference of SNR for these two samples, the precision remains similar (SD of 0.43% vs. 0.49%). This suggests that the precision of the ee% measurement is here dominated by the repeatability of the line-fitting procedure, rather than the sensitivity of the NMR experiment.

## 4. Conclusion

Enantiomers of FLX are spectrally discriminable and quantifiable by ¹⁹F NMR performed in a weakly-orienting chiral solvent. The determination of ee% can be achieved with a high level of accuracy and in a rapid NMR experiment (e.g., about 4 min). The reported method is applied without the support of any a prior calibration procedure or correction factor, while the polymer used to generate the liquid-crystalline phase is
commercially available. Interestingly, this work underlines the possibility of generating uniform and stable liquid-crystalline phases in case of hydrochloride molecules. The oriented samples are sufficiently uniform after several cycles of centrifugation to provide ¹⁹F NMR spectra with a suitable enantiomeric resolution at the day of the sample preparation. In the absence of a centrifuge, manual homogenization can be considered with comparable results, but only from three days after sample preparation.

Based on the presented ¹⁹F NMR results, the range 1–10 mg (i.e., 8.0–72.2 mmol/L) of FLX hydrochloride (molecular weight = 345.8 g/mol) is optimal in terms of enantioresolution and sensitivity for few minutes of NMR experimentation. In this range, the difference of ¹⁹F–¹⁹F total coupling between enantiomers can reach 53 Hz at 26.9 °C, which offers a comfortable enantiomeric resolution even with routine or even low-field benchtop NMR spectrometers as the magnitude of the dipolar coupling interaction is independent of the magnetic field strength.

From a practical aspect, chiral anisotropic ¹⁹F–{¹H} NMR applied to fluorinated APIs has several advantages: i) recording 100% abundant nuclei with a high gyromagnetic ratio, ii) no heteronuclear couplings increasing the distribution of peaks, iii) simple spectral pattern to analyze (e.g., a singlet for an isolated fluorine nucleus or a triplet for a CF₃ group), and iv) the absence of undesired signals due to the presence of non-fluorinated impurities or traces of water in the co-solvent or polymer.

From a more fundamental point of view, this work also shows the significant influence of the FLX concentration on the magnitude of the (¹⁹F–¹⁹F)-RDC in an enantiospecific manner. This parameter is therefore important for the achievable enantiomeric resolution. These variations of dipolar coupling can be regarded as a progressive change of the average orientation of FLX enantiomers according to their concentration in the chiral mesophase. The sample temperature also influences the magnitude of this coupling but in a different way with respect to the concentration range: a similar decrease of the (¹⁹F–¹⁹F)-RDC for both (R)- and (S)-FLX is observed from 23.8 to 216.3 mmol/L, while the temperature effect varies from one enantiomer to another in more diluted samples. The origins of such changes of enantiomer average orientations arise from a complex interplay between solute-polymer and solute-solute interactions that are both sensitive to temperature and solute concentration. Deciphering these complex mechanisms underlying the chiral recognition in such liquid oriented media remains an on-going challenge, for which these reported outcomes are insightful materials for a better understanding.

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Figures captions

**Fig. 1.** Molecular structure of (A) poly-γ-benzyl-L-glutamate (PBLG), (B) 2,2,2-trifluoro-1-phenylethan-1-ol (TFPE), and (C) fluoxetine (FLX) hydrochloride. The molecular weights (MWs) of PBLG monomer, TFPE and FLX are given below. The asterisks indicate the stereogenic centers.

**Fig. 2.** 282.4 MHz ¹⁹F–{¹H} nuclear magnetic resonance (NMR) spectra of (rac)-2,2,2-trifluoro-1-phenylethan-1-ol (TFPE) (A) and (rac)-fluoxetine (FLX) hydrochloride (B) recorded at 26.9 °C in chiral anisotropic poly-γ-benzyl-L-glutamate (PBLG)/CHCl₃ environment (top) and isotropic CHCl₃ solution (bottom). The T<sub>FF</sub><sup>(1)</sup> and T<sub>FF</sub><sup>(2)</sup> measured (absolute values) are equal to 111.8 and 73.3 Hz, respectively. The Δδ<sup>(1)</sup> and Δδ<sup>(2)</sup> variations are 0.361 and 0.200 ppm, respectively. The R/S assignment of enantiomers in the FLX racemic mixture was achieved by comparison with a scaleic sample of FLX. The T<sub>FF</sub><sup>(R)</sup> and T<sub>FF</sub><sup>(S)</sup> measured (absolute values) are equal to 55.4 and 89.5 Hz, respectively. The Δδ<sup>(R)</sup> and Δδ<sup>(S)</sup> variations are 0.042 and 0.131 ppm, respectively.

**Fig. 3.** ¹⁹F Z-imaging contour plot maps of poly-γ-benzyl-L-glutamate (PBLG)/CHCl₃ mesophases including 15 mg of fluoxetine (FLX) hydrochloride recorded the day of the sample preparation: (A) without any homogenization, (B) after 10 cycles of centrifugation procedure (500 rpm, 30 s) with nuclear magnetic resonance (NMR) tube inversion between the successive cycles, and (C) with manual homogenization based only on gravity flow of the lyotropic liquid-crystal (LLC) (no centrifugation). The one-dimensional (1D) spectra (in blue) displayed in *F*₂ projections correspond to the ¹⁹F–{¹H} 1D spectra recorded on the entire sensitive volume of the nuclear magnetic resonance (NMR) probe. Note the non-Lorentzian shape of the lines due to non-ideal LLC uniformity on Figs. 3A and C.

**Fig. 4.** Influence of the mass of fluoxetine (FLX) hydrochloride in the anisotropic ¹⁹F–{¹H} nuclear magnetic resonance (NMR) spectra. (A) Series of 282.4 MHz ¹⁹F–{¹H} NMR spectra recorded in poly-γ-benzyl-L-glutamate (PBLG)/CHCl₃ as a function of the FLX: from 20 mg (145 mmol/L) at the top, to 0.4 mg (2.9 mmol/L) at the bottom. All spectra were recorded at 26.9 °C. (B) Experimental variation of the total coupling |T<sub>FF</sub>| for both enantiomers as a function of the FLX content. The black curve shows the variation of absolute values of the difference of |T<sub>FF</sub>| between enantiomers: ||T<sub>FF</sub>(R)| – |T<sub>FF</sub>(S)||.

**Fig. 5.** Local order parameters related to a CF₃ group. (A) Visual representation of the three homotopic fluorine nuclei of the CF₃ group. (B) Example of relative positions of two F···F vectors with respect to axis *B*₀ and leading to positive and negative *S*<sub>FF</sub> values. (C)
Variation of the local order parameter $S_{FF}$, according to the trigonometric term, $(3\cos^2\theta_{FF}^{B_0} - 1)/2$ versus $\theta_{FF}^{B_0}$. $\theta_m$ is the magic angle (i.e., 54.7°) that cancels out the $S_{FF}$ value.

**Fig. 6.** Influence of the $T$ (°C) on the $^{19}$F–{¹H} nuclear magnetic resonance (NMR) spectra of fluoxetine (FLX) hydrochloride in poly-γ-benzyl-L-glutamate (PBLG)/CHCl₃. (A, B) Influence of $T$ (°C) with 15 mg of FLX: Stacked spectra recorded from 23.9 to 41.9 °C (A) and variation of $|T_{FF}(S)|$ and $|T_{FF}(R)|$ values vs. $T$ (°C) (B). (C, D) Influence of $T$ (°C) with 0.4 mg of FLX: Stacked spectra recorded from 23.9 to 41.9 °C (C) and variation of $|T_{FF}(S)|$ and $|T_{FF}(R)|$ values vs. $T$ (°C) (D). The black curve corresponds to $||T_{FF}(R)| - |T_{FF}(S)||$.

**Fig. 7.** Superimposed experimental (brown) and reconstructed (pink) $^{19}$F–{¹H} spectra after line-fitting treatment. The inset shows a vertical zoom of the two central lines of the fluoxetine (FLX) signals with the residue curve (red) between the experimental and reconstructed spectra. The spectrum was recorded on 10 mg of (rac)-FLX hydrochloride dissolved in poly-γ-benzyl-L-glutamate (PBLG)/CHCl₃ at 26.9 °C, and enantiomeric excess (ee%) was calculated using the peak areas of two most deshielded lines (i.e., left components of triplets) of (R)- and (S)-isomers.
## Acknowledgments

The authors thank the French National Research Agency (ANR) for its financial support (Grant No.: ANR-23-CE29-0005-01) in the developments of this research. The authors also thank the National Center for Scientific Research (CNRS), France and the University of Paris-Saclay, France for their recurrent funding. They would also like to thank the Institute of Molecular Chemistry and Materials of Orsay (ICMMO), France and the University of Paris-Saclay (Mutualized regional equipment (ERM) funding) for their financial participation to the BBFO 5-mm NMR probe as part of the renewal of the technical and scientific platform. Finally, the authors acknowledge Louise Meraud who participated to this work as a Master student.
<table><caption>**Table 1.** Longitudinal relaxation times $T_1(^{19}\text{F})$ measured peak by peak for the two triplets of fluoxetine (FLX) hydrochloride enantiomers. Results obtained on a sample with 20 mg of (rac)-FLX in poly-γ-benzyl-L-glutamate (PBLG)/CHCl₃ at 26.9 °C</caption><thead><tr><th>δ <sup>19</sup>F (ppm)</th><th>Component<sup>a</sup> (d, c, s)</th><th>Assignment (R, S)</th><th>T<sub>1</sub>(<sup>19</sup>F) <sup>b</sup> (ms)</th><th>RSD <sup>c</sup> (%)</th></tr></thead><tbody><tr><td>-60.77</td><td>d</td><td>R</td><td>886.9 ± 26.2</td><td>1.5</td></tr><tr><td>-61.17</td><td>c</td><td>R</td><td>1070.0 ± 56.4</td><td>2.6</td></tr><tr><td>-61.56</td><td>s</td><td>R</td><td>1005.5 ± 37.3</td><td>1.9</td></tr><tr><td>-60.83</td><td>d</td><td>S</td><td>885.7 ± 30.8</td><td>1.7</td></tr><tr><td>-61.19</td><td>c</td><td>S</td><td>1067.4 ± 67.4</td><td>3.2</td></tr><tr><td>-61.55</td><td>s</td><td>S</td><td>981.6 ± 57.5</td><td>2.9</td></tr></tbody></table>

<sup>a</sup> d, c, s: deshielded, central, and shielded resonance of triplet. <sup>b</sup> Mean $T_1(^{19}\text{F})$ value from five successive experiments, the uncertainty corresponds to the 95% confidence interval determined here as two times the standard deviation. <sup>c</sup> Relative standard deviation (RSD) computed on the same five successive experiments.

<table><caption>**Table 2.** Enantiomeric excess (ee%) measured on racemic and scalemic fluoxetine (FLX) hydrochloride samples in poly-γ-benzyl-L-glutamate (PBLG)/CHCl₃.</caption><thead><tr><th>ee<sub>theo</sub> (S) (%)</th><th>ee<sub>exp</sub> (S)<sup>c</sup> (%)</th><th>Deviation (%)</th><th>SD<sup>d</sup> (%)</th><th>SNR (R)<sup>f</sup></th><th>SNR (S)<sup>f</sup></th></tr></thead><tbody><tr><td>0<sup>a</sup></td><td>0.09</td><td>0.09</td><td>0.08</td><td>960</td><td>931</td></tr><tr><td>8.94 ± 1.14<sup>b</sup></td><td>8.87</td><td>0.07</td><td>0.43</td><td>1524</td><td>1791</td></tr><tr><td>29.9 ± 1.14<sup>a</sup></td><td>28.1</td><td>1.84</td><td>0.49</td><td>444</td><td>734</td></tr></tbody></table>

<sup>a</sup> Total mass of FLX of 10 mg. <sup>b</sup> Total mass of FLX of 15 mg. <sup>c</sup> The experimental ee% corresponds to the mean values calculated on five successive nuclear magnetic resonance (NMR) experiments of 4 min. <sup>d</sup> Standard deviation (SD) value on 5 successive experiments. <sup>f</sup> Signal-to-noise ratio (SNR) measured on the deshielded component of the triplet.
A

PBLG

<figure><img src="image_5.png" /></figure>

MW = 176.1 g/mol

B

TFPE

<figure><img src="image_6.png" /></figure>

MW = 345.8 g/mol

C

FLX

<figure><img src="image_7.png" /></figure>

MW = 219.3 g/mol

<figure><img src="image_8.png" /></figure>
<figure><img src="image_9.png" /><figcaption>B</figcaption></figure>

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A

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B

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A

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B

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C

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A

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C

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B

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D

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<figure><img src="image_27.png" /></figure>
## Highlights:

* Enantiomeric analysis of hydrochloride fluoxetine is addressed by ¹⁹F NMR spectroscopy in a chiral orienting solvent.

* The proposed method is fast (NMR experiments lasting a few minutes), relies on a commercial polymer (PBLG) and is suitable with a non-deuterated solvent (CHCl₃).

* The achievable chiral resolution allows the presented method to be applied on routine NMR devices operating at medium magnetic fields.

* The uniformity and stability of a liquid-crystal hosting an amine-contained drug as a hydrochloride salt is established by ¹⁹F NMR imaging.

* The influence of some experimental parameters, such as solute concentration and temperature on the enantiomeric resolution is investigated and discussed in detail.
March 8th, 2025

Dr. Boris Gouilleux

Assist. Prof. (Maître de conférences)

University of Paris-Saclay,
ICMMO (UMR 8182), 17-19 avenue des Science d'Orsay
91400 Orsay, France.

Mail: boris.gouilleux@universite-paris-saclay.fr

Prof. Langchong He
Editor in Chief

Xi'an Jiaotong University School of Pharmacy, China
**Journal of Pharmaceutical Analysis**

Dear Prof. Langchong He,

The authors declare no financial/personal interests or conflicts of interest that might affect their
objectivity in this research article.

Yours sincerely,

Dr. Boris Gouilleux,
University Paris-Saclay
On behalf of co-authors.

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