Hancu Gabriel (Orcid ID: 0000-0003-2564-9271)

Achiral and chiral analysis of duloxetine by chromatographic and

electrophoretic methods, a review on the separation methodologies

Daniela Lupu, Gabriel Hancu*

Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Medicine,

Pharmacy Science and Technology of Târgu Mureş, Romania;

* Corresponding author: Gabriel Hancu, Department of Pharmaceutical Chemistry, Faculty of

Pharmacy, University of Medicine, Pharmacy Science and Technology of Târgu Mureş,
Gheorghe Marinescu 38, RO-540139, Târgu Mureş, Romania, E-mail:
gabriel.hancu@umfst.ro

Abstract:
Duloxetine (DLX) is a widely used antidepressant drug belonging to the class of selective
serotonin and norepinephrine reuptake inhibitors (SNRIs); its efficacy has been demonstrated
in the treatment of not only major depressive disorders but also diabetic neuropathic pain,
generalized anxiety disorder, fibromyalgia or stress urinary incontinence. It is a chiral
substance and is used in therapy in the form of the enantiopure S-DLX which is twice as active
as R-DLX. Several methods have been published for the achiral and chiral determination of
DLX in pharmaceuticals, biological materials and environmental samples, the majority using
liquid chromatography (LC) and capillary electrophoresis (CE) coupled with different
detection techniques (UV detection, fluorescence, mass spectrometry). The aim of the current
review is to provide a systematic survey of the analytical techniques used for the determination
of DLX from different matrices.

Keywords: duloxetine, enantiomers, chiral separation, liquid chromatography, capillary

electrophoresis

This article has been accepted for publication and undergone full peer review but has not been
through the copyediting, typesetting, pagination and proofreading process which may lead to
differences between this version and the Version of Record. Please cite this article as doi:
10.1002/bmc.4883

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Introduction
Depression is a chronic medical condition considered to be one of the most common and
prevalent psychological and behavioural disorders; it is estimated to become in 2020, the
second disease leading to disability, after coronary heart disease, according to the World Health
Organization (WHO) (Wang et al., 2019).
Being characterized by a large variety of emotional, behavioural, cognitive and physical
symptoms, depression is caused by various pathogenetic factors. Recent research in
neuropharmacology and neuroimaging, support the existing relationship between the three
monoamine neurotransmitters (serotonin, norepinephrine, dopamine) and a deviation of the
normal function of different brain circuits, involved in the regulation of specific clinical
domains in depression (LaPia, 2009). In this context, specific symptoms are correlated with the
increase or decrease of certain neurotransmitters, indicating that the symptoms of depression
could be caused by specific neurochemical processes, and that antidepressant medication
should target symptoms-specific to neurotransmitters (Nutt, 2008).
Duloxetine (N-methyl-3-(1-naphthyloxy)-3-(2-thienyl)propan-1-amine) (DLX) is a modern
antidepressant from the class of serotonin–norepinephrine reuptake inhibitors (SNRIs)
(Bymaster et al., 2005). DLX possesses a chiral centre in its structure, which leads to the
existence of two enantiomers, S-DLX and R-DLX. The chemical structures of the two
enantiomers are presented in Figure 1.
The differences of the pharmacokinetic and pharmacological profiles of DLX enantiomers are
well documented, S-DLX being a more potent inhibitor of serotonin and norepinephrine. In
therapy DLX is used in the form of pure enantiomer, S-DLX. The eutomer S-DLX is twice as
active as the distomer R-DLX, which is not used clinically (Gupta et al., 2007)
S-DLX was approved for medical use by the Food and Drug Administration (FDA) in 2004
under the commercial name Cymbalta (Eli Lilly) for the treatment of major depressive
disorders (Wong, 1998).
DLX acts through a dual mechanism of action inhibiting both serotonin and norepinephrine
reuptake (Muscatello et al., 2019). It has also been shown that DLX also increases dopamine
levels within the prefrontal cortex, through a mechanism that involves norepinephrine reuptake
pumps inhibition. The affinity of DLX for other receptors (histaminergic, adrenergic,
cholinergic) does not have a clinical significance, which can be translated into a safer profile
compared to tricyclic antidepressants (Mallinckrodt et al., 2006; Westanmo et al., 2005).
Currently the use of DLX in therapy, includes not only the treatment of major depressive
disorder, but also other pathologies, such as generalized anxiety disorder, diabetic peripheral

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neuropathic pain, fibromyalgia, chronic musculoskeletal pain, and stress urinary incontinence
(Sansone & Sansone, 2014). Recent studies show an interest in the use of DLX in patients with
chemotherapy-induced neuropathies, or chronic postsurgical pain (Bedin et al., 2017;
Farshchian et al., 2014)
DLX is acid labile and is administered as enteric coated pellets in order to avoid degradation
in the acidic stomach environment. DLX is well absorbed after oral administration, with a Cmax
occurring after 6 hours. Its absolute oral bioavailability ranges between 32-80%. It binds
approximately 96% to human plasma proteins, especially to albumin and α1-acid glycoprotein.
(Sharma et al., 2000).
DLX is extensively metabolized in liver, but major metabolites do not significantly contribute
to its pharmacological activity. DLX is metabolized via two cytochrome P450 oxidative
isoenzymes (CYP1A2 and CYP2D6) followed by conjugation to form multiple metabolites.
DLX pharmacokinetics shows high interindividual variability influenced by race, age, smoking
habits and CYP2D6 metabolizing status (Sharma et al., 2000).
The oxidation of the naphthyl ring at either the 4-, 5-, or 6- positions followed by further
oxidation, methylation, and/or conjugation represents the major biotransformation route for
DLX metabolization. The major metabolites found in plasma are the glucuronide conjugates
of 4-hydroxy DLX, 4, 6-dihydroxy DLX, 6-hydroxy-5-methoxy DLX and a sulfate conjugate
of 5-hydroxy-6-methoxy DLX. Among these metabolites, the glucuronide conjugate of 4-
hydroxy DLX is the most abundant metabolite found in plasma, followed by the sulfate
conjugate of 5-hydroxy-6-methoxy DLX. The same major metabolites are found also in urine,
together with several additional ones (Lantz et al., 2003).
After the primary oxidation at either the 4-, 5-, or 6- position of the naphthyl ring, the hydroxyl
compounds resulted can either be conjugated or they can undergo further oxidation. Therefore,
they lead to a catechol intermediate or another dihydroxy derivative. Under methylation, the
catechol may form a methyl catechol, following up sulfation and glucuronidation. A minor
pathway for 5- or 6-hydroxy DLX is the rapid formation of a dihydrodiol, from an epoxide
intermediate. Another minor pathway is represented by the cleavage of DLX at the chiral centre
to form a thienyl alcohol and naphthol (Knadler et al., 2011; Lantz et al., 2003)
A scheme of major biotransformation pathways of DLX in humans is presented in Figure 2.
DLX is eliminated through urine, its elimination half-life is between 8 - 17 hours (average 12
hours). Steady state is usually achieved after 3 days. Only trace amounts of unchanged DLX
can be found in urine, the rest being eliminated as metabolites (approximately 70%); and 20%

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is eliminated in feces. Plasma clearance after oral administration ranges between 22 – 216
L/hour (average 101 L/hour) (Knadler et al., 2011; Sharma et al., 2000).
The main strategies used in the asymmetric synthesis of S-DLX can be divided into two main
categories: chemical asymmetric reduction and chemoenzymatic method (Larik et al., 2016).
Since DLX is used therapy as a pure enantiomer, enantioselective analytical methods have been
developed to verify and ensure its optical purity. In modern therapy production of enantiopure
drugs, and evaluation of their optical purity are particularly important in the development of a
chiral compound. Chiral methods must separate the two enantiomers of DLX and detect low
amounts of enantiomeric impurities (ICH recommends that chiral methods must be able to
detect amounts of impurities below 0.1%) (Q3B, Impurities in new drug products).
The aim of the present review is to summarize the published articles regarding the achiral and
chiral analysis of DLX from different matrices.

Chromatographic methods
The most established separation techniques used in pharmaceutical analysis are LC methods
coupled with different detection systems. Enantiomer separation by LC can be performed
directly using chiral selectors integrated either in the stationary or mobile phase or indirectly
using chiral derivatization reagents to form diastereomeric derivatives (Ward & Ward, 2012).

Achiral chromatographic methods

In 1996 before DLX approval by FDA, a HPLC method was used for the determination of DLX
(LY248686) and its intermediate desmethyl-DLX in human serum. After plasma extraction
with hexane, the concentrated extract was derivatized with dansyl chloride. A Phenomenex
Primesphere 5 C18 (250 x 4.6 mm, 5 µm) column was used for the separation, followed by
fluorescence detection with excitation and emission wavelengths at 285 and 525 nm
respectively (Johnson et al., 1996).
A LC method was developed for the simultaneous analysis of three crucial intermediates in the
synthesis of S-DLX; 2-acetyl thiophene, N,N-dimethyl-3-keto-(2-thienyl)-propanamine and S-
N,N-dimethyl-3-hydroxy-(2-thienyl)-propanamine. A reversed phase LC (RP-LC) method was
applied using a LichroCART RP-18 (500 × 4.6 mm, 5 µm) column and an isocratic mobile
phase consisting of acetonitrile and 0.05 M phosphate buffer (pH 7.0) containing 0.02%
diethylamine. The method was applied to determine the transformation of 2-acetyl thiophene
in N,N-dimethyl-3-keto-(2-thienyl)-propanamine, followed by the biocatalytic transformation
of the latest in N,N-dimethyl-3-hydroxy-(2-thienyl)-propanamine. All three substances are

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main intermediates of the synthesis of S-DLX, both by chemical and chemoenzymatic methods,
thus the method can be used for routine analysis during S-DLX synthesis (Soni et al., 2005).
RP-LC was used for the determination of S-DLX and its impurities and degradation products
generated from forced decomposition studies (hydrolysis, oxidation, photolysis,
thermodegradation). DLX proved to be stable under induced stress condition, except acid
hydrolysis. A Zorbax XDB C18 (50 X 4.6 mm, 5 µm) column and a mobile phase consisting a
mixture of aqueous 0.1% trifluroacetic acid: methanol: tetrahydrofuran (60:20:20, v/v/v) was
used in the separation (Srinivasulu et al., 2008).
DLX was determined in human plasma by LC tandem mass spectrometry (LC-MS) using a
Thermo Hypersil-Hypurity C18 (150 x 2.1 mm, 5 µm) column and a single-quadrupole MS
with an electrospray interface (ESI) operated in the selected-ion monitoring mode. The method
was used in a pharmacokinetic study on healthy Chinese volunteers receiving a single oral dose
of DLX (Ma et al., 2007).
An LC with atmospheric pressure ionization (API) –tandem MS technique was used for the
quantification of DLX in human plasma. The method employed protein precipitation with
methanol for the sample preparation. Separation was performed on a Gemini-C18 (50 x 4.6
mm, 3 µm) column with a mobile phase consisting of a mixture of acetonitrile: 5 mM
ammonium acetate (45:55, v/v) (pH 3.5). The lower limit of quantification (LLOQ) was 0.1 ng
mL-1. The method was applied for the quantification of DLX in 12 healthy subjects after single
dose administration (Selvan et al., 2007).
A HPLC method with pre-column derivatization and fluorescence detection was developed for
the quantification of DLX in pharmaceutical preparations. An Inertsil C18 column (150 x 2.1
mm, 5 µm) and mobile phase consisting of methanol and water (65:35, v/v) was used in the
determination. 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole was used as derivatizing agent. The
excitation and emission wavelengths of the fluorescence detector were 461 and 521 nm,
respectively. Limits of detection (LOD) and quantification (LOQ) were 0.51 and 1.53 ng mL-1
respectively (Tatar Ulu, 2012).
Two of the major metabolites of DLX, 4-hydroxy DLX glucuronide and 5-hydroxy-6-methoxy
DLX sulfate were determined in human plasma by LC-MS. An Aquasil C18 column (150 mm
× 2 mm, 5 µm) was used in the separation; for the determination of 4-hydroxy DLX glucuronide
the mobile phase consisted of water: acetonitrile: formic acid (80:20:0.1, v/v/v) while for the
determination of 5-hydroxy-6-methoxy DLX sulfate the mobile phase was
water/acetonitrile/acetic acid (70: 30: 0.05, v/v/v). Solid phase extraction (SPE) was used for
sample preparation, positive ESI for the determination of 4-hydroxy DLX glucuronide,

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chromatographic resolution of 4-, 5-, 6-hydroxy DLX glucuronide positional isomers, and
negative ion ESI for the determination of 5-hydroxy-6-methoxy DLX sulfate. The method was
applied in pharmacokinetic studies involving DLX metabolism (Satonin et al., 2007).
An LC-MS/MS method has been developed for the quantification of DLX in human plasma.
The analyte was extracted using a liquid-liquid extraction (LLE) using methyl-tert-butyl ether:
n-hexane (80:20). An X-terra RP8 (50 x4.6 mm, 5 μm) column and an isocratic mobile phase
consisting of 30 mM ammonium formate (pH 5.0): acetonitrile was used in the separation. The
LLOD and LLOQ were 0.04 ng mL-1 and 0.1 ng mL-1 (Reddy et al., 2012).
DLX and its major metabolite 4-hydroxy-DLX glucuronide were determined in rat plasma with
an LC-MS/MS. An Atlantis C18 (50 x 4.6 mm, 3 µm) column and an isocratic mobile phase
consisting of methanol: 5 mM ammonium acetate (6:4, v/v) were used in the separation. Plasma
samples were precipitated with methanol. Quantification was performed on a triple-quadrupole
MS using ESI, and the ion transition monitored in selective reaction monitoring mode. The
method was used to measure concentrations of DLX and 4-hydroxy-DLX in rat plasma after a
single dose administration (Chae et al., 2013).
An LC-MS/MS method was used for the determination of DLX in human plasma. Solid phase
extraction (SPE) was used for sample preparation. A Zorbax SB C18 (50 x 2.1 mm, 5 µm)
column and a mobile phase consisting of acetonitrile: 5 mM ammonium acetate buffer (83:17,
v/v) was used in the separation. A short migration time of 2.5 minutes makes this method
attractive for the rapid bioanalysis of DLX (Gajula et al., 2013).
DLX was determined also by LC-UV in human plasma in the presence of its major metabolite,
4-hydroxy-DLX glucuronide. An Orbit 100 C18 (250 × 4.6 mm, 5 μm) column and a mobile
phase consisting of 0.02 M sodium dihydrogen phosphate buffer (pH 3.0): acetonitrile (62:38,
v/v) was used in the separation. No evidence regarding possible conversion of the major
metabolite into the parent substance was found. The method can be applied in pharmacokinetic
studies (Kaza et al., 2014).
An LC-MS/MS method was used for the simultaneous quantification of quetiapine (second-
generation antipsychotic) and DLX in rat plasma. A one-step protein precipitation with
acetonitrile was used for sample preparation. An Eclipse XDB-C18 (250 × 4.6 mm, 5 μm)
column and a mixture of acetonitrile and 2 mM ammonium formate containing 0.1% formic
acid at a gradient elution was used in the separation. The triple quadrupole MS was operated
in positive ion ESI mode, and a multiple reaction monitoring mode was applied for the
quantification. Pharmacokinetic interaction of quetiapine and duloxetine was studied; it was

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found that concomitant administration of both substances significantly increased plasma
concentrations of DLX (Chen et al., 2019).
DLX was detected in post-mortem cases using gas chromatography – mass spectrometry (GC-
MS). A liquid-liquid extraction (LLE) procedure using n-buthylchloride was used for sample
preparation. Gas chromatography-nitrogen-phosphorus detection (GC-NPD) technique proved
to be less sensitive for the detection of DLX. DLX was not reported as the primary cause of
death, based on the results of toxicological and autopsy report; however, DLX has a high
distribution volume and exhibits post-mortem redistribution as expected (Anderson et al.,
2006).
The analytical parameters of the LC achiral analysis methods for the analysis of DLX are
summarized in Table 1.

Chiral chromatographic methods

DLX enantiomers were separated using direct HPLC methods employing either
hydroxypropyl-β-CD (HP-β-CD) as chiral selector added to the mobile phase or a Chirobiotic
V (200 × 4.6 mm, 5 µm) vancomycin based chiral column, respectively. Both methods resulted
in the baseline separation of the two enantiomers. The method using the chiral additive
employed an Agilent XDB-C8 achiral column (200 × 4.6 mm, 5 µm) and a mobile phase
composed of 30 mM phosphate buffer (pH 2.0) containing 60 mM HP-β-CD: methanol (65:35,
v/v). The method using the chiral stationary phase (CSP) used a mobile phase containing
methanol: acetic acid: triethylamine (100:0.04:0.01, v/v/v) and was chosen to determine the
enantiomeric purity of S-DLX, based on the shorter analysis time and higher chiral resolution
(1.7 resolution and 20 minutes analysis time vs. 1.3 resolution and 60 minutes analysis time).
However, in both methods, the enantiomeric impurity, R-DLX, was the second to elute, making
it hard to be detected in the presence of high amounts of S-DLX. The LOD for R-DLX was
0.06 µg mL-1 (Yang et al., 2007).
Another direct chiral separation method for DLX enantiomers used a Chiralpak AD-H (250 ×
4.6 mm, 5 µm) amylose based chiral column and a mobile phase consisting of n-hexane:
ethanol: diethylamine (80:20:0.2, v/v/v). The resolution between enantiomers was higher than
2.8 and migration time was approximately 7 minutes. The method was applied for the
determination of enantiomeric purity of S-DLX in bulk substance; the order of migration was
the convenient one, the enantiomeric impurity, R-DLX migrating first. LOD and LOQ for R-
DLX was 250 ng mL-1 and 750 ng mL-1 respectively. The method can be useful also for
preparative HPLC separation (Rane et al., 2008).

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The enantiomers of DLX were also separated on a Chiral-AGP (150 × 4.0 mm, 5 µm) α-acid
glycoprotein chiral column using a mobile phase consisting of a 10 mM acetate buffer (pH
3.8): acetonitrile (93: 7, v/v). The resolution between enantiomers was higher than 2.2 and
migration time was approximately 8 minutes. LOD and LOQ for R-DLX was 150 ng mL-1 and
450 ng mL-1 respectively. The method was applied for the determination of S-DLX
enantiomeric purity in bulk drug and pharmaceutical preparations (Davadra et al., 2011).
The analytical parameters of the LC chiral analysis methods for the analysis of DLX are
summarized in Table 2.

Electrophoretic methods
The use of capillary electrophoresis (CE), in the of pharmaceutical substances, presents several
advantages related with the method simplicity, high efficiency, versatility, rapid analysis time,
high-resolution power, small sample volume and reagents consumption and relatively low
operating costs. Furthermore, in chiral CE usually a direct method of separation is used by
simply adding the chiral selector into the background electrolyte (Ward & Ward, 2012).

Achiral electrophoretic methods

CE with laser-induced fluorescence (LIF) detection was used for the determination of DLX in
human plasma. The analyte was derivatized with the fluorescent derivatization agent, 5-(4,6-
dichlorotriazinyl) aminofluorescein at pH 11. An LLE procedure with a mixture of hexane: 2-
propanol was used for sample preparation. The buffer solution consisted of 40 mM borate
buffer at pH 10.3, 10 mM tetrabutylammonium bromide and 10% acetone (v/v). The LOQ of
the method was 2.5 ng mL-1, and the linearity range covers therapeutic concentrations of DLX.
The method was applied for the quantification of DLX in patients undergoing antidepressant
treatment (Musenga et al., 2009).
Micellar electrokinetic capillary chromatography (MEKC) was used for the quantitative
determination of DLX and 1-naphtol. 1-naphtol is a toxic impurity obtained by degradation of
DLX in acidic medium. The buffer electrolyte contained 50 mM Tris and 20 mM SDS at pH
10.6. The LOD and LOQ were 0.50 and 1.45 µg mL-1 for DLX and 0.20 and 0.55 µg mL-1 for
1-naphtol. The method can be applied for DLX routine analysis to ensure that 1-naphtol
impurity does not surpass allowable quantities (Wingert et al., 2011).

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Chiral electrophorectic methods
The first CE method for the chiral determination of DLX (LY248686) was published long
before its FDA approval in 1994. HP-β-CD was used as chiral selector and the effects of several
analytical conditions (buffer pH and concentration, CD type and concentration) on the
separation was investigated during the method optimization process. The method was used to
determine the enantiomeric purity of the S-enantiomer. The results demonstrated that CE
separations can be an alternative to chiral HPLC separations, with advantages related to the
shorter analysis time, lower cost and higher resolution (Rickard & Bopp, 1994).
DLX was used as model compound together with other 39 compounds in a systematic CD
screening approach for the enantiodetermination of small amine-containing chiral substances.
A 30 mM phosphate buffer at pH 2.5 was used in the determination and dimethyl-β-CD (DM-
β-CD), hydroxypropyl-α-CD (HP-α-CD), hydroxypropyl-β-CD (HP-β-CD), hydroxypropyl-γ-
CD (HP-γ-CD) and sulfated-β-CD (S-β-CD) were tested as chiral selectors. Chiral resolution
was obtained in the case of DLX when using DM-β-CD, HP-α-CD, HP-β-CD and S-β-CD, and
the best resolution was obtained with 30 mM HP-α-CD (Liu & Nussbaum, 1999).
In another study evaluating the potential use of erythromycin lactobionate as chiral selector in
non-aqueous capillary electrophoresis (NACE) in methanol-based medium, DLX was chosen
as a model basic chiral compound together with propranolol. The experiments used a Tris-boric
acid buffer and methanol as the organic medium. The effects of several analytical conditions
(organic solvent type, buffer pH and composition, chiral selector concentration, capillary
temperature, applied voltage) on the separation was investigated. Baseline separation of DLX
was obtained but with long migration times, approximately 50 minutes; demonstrating
potential in using macrocyclic antibiotics in NACE chiral separations (Chen B et al., 2010).
DLX was used as model compound together with other 8 basic chiral substances to evaluate
the use of glycogen-based polysaccharides in dual chiral CE system. Three glycogen-based
dual chiral CE systems consisting of glycogen (neutral polysaccharide) and chondroitin sulfate
A (ionic polysaccharide), β-CD and HP-β-CD were tested; the combination between glycogen
and chondroitin sulfate A led to best results. The enhancement of enantioseparation when using
the glycogen based dual system by comparison with single selector system was observed,
which indicates synergistic effects of the selectors probably because of some favourable
interaction effects between glycogen and chondroitin sulfate A (Chen J et al., 2010).
Two CE chiral methods with UV and MS detection was used to verify the enantiomeric purity
of DLX. In the CE-UV experiment the best results were obtained when using 150 mM
phosphate buffer at pH 3.0 and 0.5% (w/v) HP-β-CD as chiral selector; while for CE-MS with

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electrospray ionization (ESI) a 150 mM ammonium formate buffer at pH 3.0 and 0.5% (w/v)
HP-β-CD as chiral selector was used. Both methods resulted in the baseline chiral resolution
of DLX with resolution of over 2.0, the enantiomeric impurity R-DLX migrated first. The LOD
of the method was 200 ng mL-1 in the case of CE-UV method and 20 ng mL-1 in the case of
CE-MS method, allowing identification of 0.02% R-DLX enantiomeric impurity. The method
was applied for the analysis of pharmaceutical formulations (Sanchez‐Lopez et al. (1), 2014).
A complex screening involving 15 CD derivatives was carried out in order to establish the
optimum chiral selector for the enantioresolution of DLX. The best resolution were obtained
when using HP-β-CD or methyl- γ-CD (M-γ-CD); the enantiomer migration order varied
depending on the CD; R-DLX migrating first in the case of HP-β-CD and second in the case of
M-γ-CD. Nuclear magnetic resonance spectroscopy (NMR) and MS experiment were carried
out to characterize DLX-CD complexes, including their averaged stoichiometry, their
thermodynamic apparent and averaged equilibrium constants. NMR results indicated that HP-
β-CD formed a more stable complex with R-DLX than with S-DLX, and S-DLX binds more
strongly to M-γ-CD than R-DLX; in contradiction with migration order obtained in CE.
Therefore, it seems DLX chiral separation by CE relies not only on the thermodynamic stability
of the DLX-CD complexes but also on their electrophoretic mobility (Sanchez‐Lopez et al. (2),
2014).
DLX was used as model compound along with other three substances (citalopram,
ketoconazole, sulconazole) in a study of dextrin-based synergistic systems with chiral ionic
liquids (CIL) as additives for enantiomeric separation in CE. It was found that the use of a
CILs-dextrin synergistic system led to improved separation and selectivity compared with
single dextrin system. In order to optimize enantiomeric separation with the CILs-dextrin
system, the influence of key parameters such as CIL concentration, dextrin concentration,
buffer pH and composition and applied voltage was investigated (Zhang et al., 2019).
A stability and toxicity study of DLX and econazole racemic mixture and individual
enantiomers was developed under abiotic and biotic conditions. Toxicity was evaluated on the
aquatic plant Spirodela polyrhiza. A 25 mM phosphate buffer at pH 3.0 and 1.5% S-β-CD as
chiral selector was used in the experiment, leading to the simultaneous chiral separation of both
enantiomers in approximately 8 minutes. Stability evaluation showed different decay
percentages for both individual substances and their binary mixtures. Calculated EC50 values
of 0.81 mg L-1 for DLX and 0.44 mg L-1 for econazole and their binary mixtures of 0.69 mg L-
1
allowed both drugs to be included in the category of toxic compounds (Valimaña-Traverso et
al. (1), 2019).

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The same authors published another stability and toxicity study of DLX and econazole using a
freshwater organism belonging to the microcrustacean family, Daphnia magna. A CE method
using 25 mM phosphate buffer at pH 3.0 and 1.5% S-β-CD as chiral selector was used for
quantification of the analytes. Differences in DLX concentrations were negligible in single
solutions, whereas the stability profiles of DLX racemic mixtures and enantiomers varied in
mixtures of both drugs after 72 hours (Valimaña-Traverso et al. (2), 2019).
The analytical parameters of the CE chiral analysis methods for the analysis of DLX are
summarized in Table 3.

Conclusions
Current regulations on chiral drugs pressed the research activity towards the development,
optimization and validation of new, fast and reliable analytical methods for the determination
of optically active pharmaceutical substances from pharmaceutical formulation or in complex
matrices like biological fluids.
Analytical methods used for the achiral and chiral determination of DLX include mainly LC
and CE techniques. Clearly, the main field of chromatographic and electromigration methods
is separation on an analytical scale for the regulation of enantiomer purity in synthesis,
racemization processes testing, pharmaceutical quality control and pharmacokinetic studies.
In the last 15 years several analytical methods have been published for the analysis of DLX
from different matrices (bulk substances, pharmaceutical preparations, biological fluids,
environmental samples).
A few methods are available for the determination of DLX in biological fluids, including LC
with single-quadrupole mass spectrometry (LC–MS), LC with tandem mass spectrometry (LC-
MS/MS), LC with atmospheric pressure ionization–tandem MS, gas chromatography–mass
spectrometry (GC–MS), HPLC and CE.
Direct LC enantioseparation of DLX was reported using chiral stationary phases based on
macrocyclic antibiotic (vancomycin – Chirobiotic V column), amylose (Chiralpak AD-H
column) and glycoprotein (Chiral-AGP column). In another direct enantioseparation method
HP-β-CD was used as chiral additive in the mobile phase.
DLX is a basic drug whose enantiomers will migrate in CE towards the cathode in an acidic
buffer interacting with neutral CDs. DLX enantioseparation by CE was reported in several
studies using HP-β-CD as chiral selector and UV or MS detection. Also, DLX was used as
model compound for the evaluation of glycogen-based selectors, erythromycin lactobionate or
CILs as chiral selectors in CE.

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While chromatographic methods have proven to be the most effective and flexible analytical
techniques for stereochemical analysis, CE has become a viable alternative for certain
applications due to its high efficiency, short analysis time and sample and reagent consumption
economy.

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Table 1 Chromatographic methods for the achiral determination of DLX

Chromatographic Analytical conditions Sample Analytes References

method
LC-LIF column: Phenomenex Primesphere 5 C18 (250 x 4.6 mm, 5 µm), human plasma DLX, Johnson et al.,
mobile phase: 0.05 M ammonium acetate: acetonitrile (21:79), desmethyl- 1996
flowrate: 2 mL min-1, derivatization with dansyl chloride, DLX
fluorescence detection
RP-LC column: LichroCART RP-18 (500 × 4.6 mm, 5 µm), mobile phase: bulk substance DLX, three Soni et al.,
acetonitrile: 0.02 M phosphate buffer (pH 7) containing 0.02% synthesis 2005
(w/v) diethylamine – gradient elution, flowrate: 1 mL min-1, UV intermediates
detection 241 nm
RP-LC column: Zorbax XDB C18 (50 X 4.6 mm, 5 µm), mobile phase: bulk substance DLX, Srinivasulu et
0.1% trifluroacetic acid: methanol: tetrahydrofuran (60:20:20, degradation al., 2008
v/v/v), flowrate: 0.8 mL min-1, 25 0C, UV detection 230 nm product
LC-MS column: Thermo Hypersil–Hypurity C18 (150 × 2.1 mm, 5 μm), human plasma DLX Ma et al., 2007
mobile phase: acetonitrile: methanol: 20 mM ammonium acetate pH
3.5 (42:20:38, v/v/v), flowrate: 0.24 mL min-1, 40 0C
single-quadrupole MS with selected-ion monitoring (SRM)
LC-MS/MS column: Gemini-C18 (150 × 2.1 mm, 5 μm), mobile phase: human plasma DLX Selvan et al.,
acetonitrile; 5 mM ammonium acetate (45:55, v/v, pH 3.5), flowrate: 2007
0.3 mL min-1, 20 0C
tandem mass spectrometry – API operated in positive ion mode
LC-MS/MS column: Aquasil C18 column (150 mm × 2 mm, 5 μm), mobile phase: human plasma DLX, 4- Satonin et al.,
water: acetonitrile: formic acid (80/20/0.1, v/v/v) (4-hydroxy DLX hydroxy DLX 2007
glucuronide); water/acetonitrile/acetic acid (70/30/0.05, v/v/v) (5- glucuronide, 5-
hydroxy-6-methoxy DLX sulfate), flowrate: 0.3 mL min-1, 22 0C hydroxy-6-
tandem mass spectrometry – ESI positive ion mode (4-hydroxy DLX methoxy DLX
glucuronide), negative ion mode (5-hydroxy-6-methoxy DLX sulfate
sulfate)

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LC-MS_MS column: X–terra RP8 (50 x 4.6 mm, 5 µm); mobile phase: human plasma DLX Reddy et al.,
acetonitrile–30 mM ammonium formate (90:10, v/v) (pH 5.0); 2012
flowrate: 0.4 mL min-1; 40 0C
triple-quadrupole mass spectrometry with multiple-reaction
monitoring (MRM)
LC-LIF column: Inertsil C18 column (150 x 2.1 mm, 5 µm), mobile phase: pharmaceutical DLX Tatar Ulu,
methanol: water (65:35, v/v), flowrate: 1.2 mL min-1, 40 0C, preparation 2012
derivatization 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole, fluorescence
detection
LC-MS/MS column: Atlantis C18 (50 x 4.6 mm, 3 µm), mobile phase: methanol: rat plasma DLX, 4- Chae et al.,
5 mM ammonium acetate (6:4, v/v) – isocratic elution, flowrate: 0.4 hydroxy-DLX 2013
mL min-1, 40 0C glucuronide
triple-quadrupole mass spectrometer using ESI
LC-MS/MS column: Zorbax SB C18 (50 x 2.1 mm, 5 µm), mobile phase: human plasma DLX Gajula et al.,
acetonitrile: 5 mM ammonium acetate buffer (83:17, v/v), flowrate: 2013
0.9 mL min-1, 40 0C
triple-quadrupole mass spectrometry with multiple-reaction
monitoring (MRM)
LC-UV column: Orbit 100 C18 (250 × 4.6 mm, 5 μm), mobile phase: 0.02 M human plasma DLX, 4- Kaza et al.,
sodium dihydrogen phosphate buffer (pH 3.0): acetonitrile (62:38, hydroxy-DLX 2014
v/v), flowrate: 1 mL min-1, 40 0C, UV detection 230 nm glucuronide
LC-MS/MS column: Eclipse XDB-C18 (250 × 4.6 mm, 5 μm), mobile phase: rat plasma DLX, Chen et al.,
acetonitrile: 2 mM ammonium formate containing 0.1% formic – quetiapine 2019
gradient elution, flowrate: 0.6 mL min-1, 40 0C
tandem mass spectrometry

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Table 2 Chromatographic methods for the chiral determination of DLX enantiomers

Method Analytical conditions CSP/CMPA Sample Analytes References

LC-UV mobile phase: methanol: acetic acid: triethylamine Chirobiotic V bulk substance DLX 21
(100:0.04:0.01, v/v/v); flow rate: 1.0 mL min , (150 x 4.6 mm, 5
-1

temperature 170C; UV detection 214 nm µm) –

vancomycin CSP,
LC-UV achiral column: Agilent XDB-C8 (200 x 4.6 mm, 5 µm); HP-β-CD - bulk substance DLX 21
mobile phase: 30 mM phosphate buffer (pH 2.0) + 60 CMPA
mM HP-β-CD: methanol (65:35, v/v); flow rate: 1.0 mL
min-1, temperature 200C; UV detection 214 nm
LC-UV mobile phase: n-hexane–ethanol–diethyl amine Chiral AD-H (250 bulk substance DLX 22
(80:20:0.2, v/v/v); flow rate: 1.0 mL min-1, temperature × 4.6 mm, 5 µm)
300C; UV detection 254 nm – amylose CSP
LC-UV mobile phase: 10 mM acetate buffer (pH 3.8): Chiral AGP (150 bulk DLX 23
acetonitrile (93:07, v/v); flow rate: 1.0 mL min-1, X 4.0 mm, 5 µm) substance,
temperature 200C; UV detection 222 nm – glycoprotein pharmaceutical
CSP s

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Table 3 Electrophoretic methods for the chiral determination of DLX enantiomers

Method Analytical conditions Chiral selectors Sample Analytes References

CE-UV 100 mM Tris-phosphate, pH 2.35, 22 kV; UV detection 4 mM HP-β-CD bulk DLX Rickard &
210 nm substance Bopp, 1994
CE-UV 30 mM phosphate/TBA; pH 2.5; +30 kV; 20 0C; UV detection 30 mM HP-α-CD bulk DLX, model Liu &
214 nm substance compounds Nussbaum,
1999
CE-UV 50 mM Tris – 150 mM boric acid, pH 2.5, +15 kV; 16 100 mM bulk DLX Chen B et
0
C; UV detection 250 nm erythromycin (in substance al., 2010
methanol)
CE-UV 40mM phosphate, pH 2.5, +25 kV; 26 0C; UV detection glycogen (3%, bulk DLX Chen J et al.,
250 nm w/v) + substance 2010
chondroitin
sulfate A (2%,
w/v)
CE-UV 150 mM phosphate; pH 2.5; +30 kV; 20 0C; UV 0.5% HP-β-CD pharmaceuti DLX (1) Sanchez-
detection 220 nm cal products Lopez et al.,
2014
CE-MS 150 mM ammonium formate; pH 3.0; +30 kV; 15 0C 0.5% HP-β-CD pharmaceuti DLX (1) Sanchez-
cal products Lopez et al.,
2014
CE-UV 50 mM phosphate; pH 2.5; +15 kV; 20 0C; UV detection 220 1% HP- β-CD bulk DLX (2) Sanchez-
nm substance Lopez et al.,
2014

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Figure 1 Chemical structures of DLX enantiomers

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Figure 2 DLX metabolism

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