PART-[A]

ENANTIOSELECTIVE METHOD

DEVELOPMENT AND VALIDATION

OF SOME PHARMACETUICALS

SECTION-2

Enantioselective HPLC Method

Development and Validation of Zopiclone

Zopiclone Section-2

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[1] Introduction

Pharmaceutical with an asymmetric carbon (chiral center) are often referred to as

chiral drugs. Chiral drugs were mainly presented as the racemate, which is mixture of

equal amounts of left and right-handed enantiomers. In an achiral environment, each

enantiomers of racemate shows completely identical physical and chemical properties.

However, in a chiral environment such as in vivo, they demonstrate different chemical,

bio-chemical, and pharmacological behaviors. In principle, therefore enantiomers in a

racemic drug should be treated as two different compounds. One enantiomer of a drug

may have a desired beneficial effect, while the other may cause serious and undesired

side effects, or sometimes even beneficial but entirely different effects [1]. Although

single-enantiomer drugs have been thought to be preferable to racemic drugs, most chiral

drugs were developed as racemates due to the lack of technologies that produce single-

enantiomers until recently. Current technologies of asymmetric synthesis and chiral

separation made it possible for pharmaceutical companies to develop single-enantiomer

drugs. Lately, many single-enantiomer drugs have been approved and marketed broadly.

Stereoisomers usually require specialized chiral techniques for their correct

identification, characterization, separation and measurement. They are often readily

distinguished by biological systems, however, and may have different pharmacokinetic

properties (absorption; distribution, biotransformation. and excretion) and quantitatively

or qualitatively different pharmacologic or toxicological effects. Approximately, 25 % of

commercially available chiral drugs are either racemates or diastereomers and biological

activity of chiral molecule substantially differs from its racemates or its stereoisomers is

well documented [2, 3]. The chiral chromatography for enantiomeric separation is now

well established procedure in the pharmaceutical industry. The direct separation of

enantiomers using columns containing immobilized chiral stationary phases (CSPs) is

usually the method of choice. Recent developments in high performance liquid

chromatography (HPLC) chiral stationary phase technology provide a more efficient and

reliable solution to analyze chiral drugs.

Zopiclone Section-2

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1.1 Description

Zopiclone has single chiral center and chemically known as ((RS)-[8-(5-

chloropyridin-2-yl)- 7-oxo-2,5,8-triazabicyclo [4.3.0]nona-1,3,5-trien-9-yl] 4-

methylpiperazine-1-carboxylate). (Fig.1). Zopiclone also marketed as a pure S(+)-

enantiomers, known as a eszopiclone. The chemical name of eszopiclone is (+)-(5S)-6-

(chloropyridine-2-yl)-7-oxo-6,7-dihydro-5H-[yrrolo[3,4-b] pyrazin-5-yl 4-

methylpiperazine-1-carboxylate.

Zopiclone is a non-benzodiazepine hypnotic agent and classed as a

cyclopyrrolone derivative, and it is used in the treatment of insomnia, where sleep

initiation or sleep maintenance are prominent symptoms. Its molecular formula is

C17H17CIN6O3 having molecular weight 388.8 g/mol. The CAS number of zopiclone is

43200-80-2.

(S)-Zopiclone (R)-Zopiclone

Figure 1: Zopiclone enantiomers

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1.2 Indication

Zopiclone is used in the treatment of insomnia, where sleep initiation or sleep

maintenance are prominent symptoms.

1.3 Mechanism of Action

Zopiclone exerts its action by binding on the benzodiazepine receptor complex

and modulation of the GABAAZ receptor chloride channel macromolecular complex.

Both zopiclone and benzodiazepines act indiscriminately at the benzodiazepine binding

site on α1, α2, α3 and α5 GABAA containing receptors as full agonists causing an

enhancement of the inhibitory actions of GABA to produce the therapeutic (hypnotic and

anxiolytic) and adverse effects of zopiclone.

1.4 Pharmacodynamics

Zopiclone is a non-benzodiazepine hypnotic from the pyrazolopyrimidine class

and is indicated for the short-term treatment of insomnia. While Zopiclone is a hypnotic

agent with a chemical structure unrelated to benzodiazepines, barbiturates, or other drugs

with known hypnotic properties, it interacts with the gamma-aminobutyric acid-

benzodiazepine (GABABZ) receptor complex. Subunit modulation of the GABABZ

receptor chloride channel macromolecular complex is hypothesized to be responsible for

some of the pharmacological properties of benzodiazepines, which include sedative,

anxiolytic, muscle relaxant, and anticonvulsive effects in animal models. Zopiclone binds

selectively to the brain alpha subunit of the GABAA omega-1 receptor.

1.5 Pharmacology

Zopiclone exhibits in-vivo chiral inversion, and virtually all sedative and hypnotic

activity of racemic zopiclone is attributable to the dextrorotatory isomer, which is

approximately twice as active as the racemate. The levorotatory isomer is both, i.e.

almost inactive and more toxic than racemic zopiclone. In mice, for example, racemic

zopiclone processes toxicity (LD50) in the region of 850 mg/kg, while the dextrorotatory

isomer, S(+)-Zopiclone, has a toxicity in the region of 1.5 g/kg, and the levorotatory

isomer, R(-)-zopiclone, processed as LD50 between 300 and 900 mg/kg. [4].

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1.6 Metabolism

The major metabolites of zopiclone are summarized in Table 1.

S.N. Substrate Enzymes Product

1 Zopiclone Cytochrome P450 2C9 Zopiclone N-oxide

2 Zopiclone Cytochrome P450 2C9 N-Desmethylzopiclone

3 Zopiclone Prostaglandin G/H

synthase 1 CO2

4 Zopiclone Cytochrome P450 2C8

Cytochrome P450 3A4 Zopiclone-N-oxide

5 Zopiclone Cytochrome P450 2C8

Cytochrome P450 3A4 N-desmethylzopiclone

Table 1: Common metabolites of Zopiclone

1.7 Adverse Reaction

The side effect most commonly seen in clinical trials is taste alteration or

dysgeusia (bitter, metallic taste, which is usually fleeting in most users but can persist

until the drug's half-life has expired). Palpitations may occur in the daytime following

withdrawal from the drug after prolonged periods of use (especially when taken for more

than two weeks). Impairment of driving skills with a resultant increased risk of road

traffic accidents is probably the most important side effect. This side effect is not unique

to zopiclone but also occurs with other hypnotic drugs [5, 6].

1.8 Macrocyclic Glycopeptide Based Chiral Stationary Phase

The macrocyclic antibiotics represent new class of chiral selectors in separation

science. This type of CSPs had an immediate and significant impact on the field of

separation science since their introduction in 1994 [7]. Glycopeptide based CSP has high

enantioselectivity properties are due to their amphoteric character, their molecular

structure that in solution accentuates the enantioselective interaction, and their

hydrophilic and hydrophobic groups, which make these groups soluble in aqueous and

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organic solvents [8]. Macrocyclic glycopeptide CSPs possess several characteristic that

allow them to interact with analytes and serve as chiral selectors. They have a number of

stereogenic centers and functional groups, allowing them to have multiple interactions

with chiral molecules. They typically have molecular masses between 600 and 2200 and

often have numerous functional groups. They may be acidic, basic or neutral and may

have little or no UV-VIS absorbance.

The first macrocyclic glycopeptide antibiotic introduced as a commercial CSP

was vancomycin (Chirobiotic V), followed by teicoplanin (Chirobiotic T), rostocetin A

(Chirobiotic R) and aglycon part of teicoplanin (Chirobiotic TAG).

The present study based on chiral method development using Chirobiotic TAG

column. Chirobiotic TAG is made by covalently bonding the aglycon part of teicoplanin

to silica gel via linkage chains (Fig. 2). It consists of the four fused macrocyclic rings,

which form a semi-rigid basket. One of the major structural characteristics of teicoplanin

is that it has a hydrophobic acyl side chain.

Figure 2: Chemical structure of Chirobiotic TAG

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The macrocyclic glycopeptide CSP can interact by any of the following

mechanism [9], depending on the analyte property and mobile phase combination [10];

1. Hydrogen bonding (strong)

2. dipole-dipole interactions (medium strong)

3. π- π interaction (strong)

4. Steric interactions (weak)

5. Inclusion (weak)

6. Anionic or cationic binding (very strong)

The shallow pockets for inclusion yield weaker binding energies when compared

to cyclodextrins. Reverse phase conditions favor inclusion and hydrogen bonding, and

under these conditions, change in pH can produce cationic or anionic interactions. The π-

π complexation and dipole stacking are favored in normal phase solvents. The unique

structure of the macrocyclic glycopeptides and their abundance of functionality (e.g.

aromatic, hydroxyl, amine, and carboxylic acid moieties, amide, linkages, hydrophobic

pockets,) give them broad selectivity for a wide variety of anionic, neutral, and cationic

compounds.

The use of methanol, ethanol, alone or in combination is useful mobile phase and

known as a polar organic mode. The addition of small amount of acid and base or volatile

salt considered as a polar ionic mode. The new polar ionic mode enhances the potential

interaction, i.e. ionic and it is useful when the compound has an ionizable group. The

column manufacture recommends the common mobile phases with respect to the analyte

properties. The details are summarized in Table 2.

CHIROBIOTIC TAG (Teicoplanin Aglycon)

Compound

Type

Polar Organic

Mode

Polar Ionic

Mode

Reversed-Phase

Mode

Normal Phase

Mode

Acids

Bases

Neutrals

Table 2: Mobile phase selection guide for Chirobiotic TAG

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[2] Literature Overview

The literature reviews regarding zopiclone suggest that various analytical methods

were reported for drug substance as well as pharmaceutical formulations. There is no

published report for stability indicating enantioselective estimation methods for

zopiclone.

Numerous analytical methods were reported for the enantiomeric separation or

quantification of zopiclone in the biological matrices using β-cyclodextrin bonded phase,

AGP and cyclobond I based chiral columns [11-15]. The drug was studied as a racemic in

biological matrices using HPLC-UV [16-18], GC [19], and LC/MS/MS techniques [20-

23]. In human plasma the degradation and racemization of zopiclone increased with

increasing the pH and temperature, and found hydrolysis of the carbamate function and

opening of the pyrrolidone ring [24, 25]. The achiral stability indicating HPLC method

has previously reported for the determination of racemic zopiclone in tablets [26, 27].

B. Koppenhoefer, U. Epperlein, B. Christian, B. Lin, Y. Ji, Y. Chen have

reported separation of enantiomers of zopiclone by capillary electrophoresis using III. β-

cyclodextrin as chiral solvating agent. The selectivity factor achieved could achieved was

1.0. [26].

Stavroula Piperaki, Maria Parissi-Poulou have reported a separation of

zopiclone enantiomers and its degradation product and chiral metabolites using β-

cyclodextrin bonded-phase CSP. The retention time of the enantiomers are 39 and 43 min

with resolution of 1.79 [28].

Sanagaraju S, Lakshmi Kanth M, Rao BM, Someswararao N. have reported

enantiomeric separation of zopiclone enantiomers in bulk drug samples using

polysaccharide based CSP. The resolution value could achieve to 1.6 using Chiracel OD-

RH column in reverse phase mode. The developed method was validated [29].

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[3] Aim of Present Study

Zopiclone was first approved as a racemic mixture, but the (S)-enantiomer

(Eszopiclone) was introduced to the market in 2005. Recent study shows (S)-(+)-

enantiomer has more than 50 times higher affinity toward the benzodiazepine receptor

binding site than (R)-(-)-enantiomer [30]. In light of the increased awareness concerning

biologically important isomers, the US Food and Drug Administration has issued certain

guidelines for the marketing of racemic compounds [31]. This new trend resulted in the

determination of chiral impurities at a concentration below 0.1 % and this places heavy

demands on the chiral analytical methods [32, 33].

Numerous analytical methods were reported for the enantiomeric separation or

quantification of zopiclone. However, to the best of our knowledge, no report has been

published on stability indicating chiral HPLC method to estimate the enantiomeric purity

of zopiclone using macrocyclic glycopeptide chiral stationary phases in the

pharmaceutical formulations. The objective of the present study was to develop a chiral

HPLC method to determine zopiclone enantiomeric purity in drug substance and drug

product using glycopeptide stationary phase. The published chiral method has runtime of

almost 50 min, so another important objective of this research was to achieve the

enantiomeric separation of zopiclone in comparatively less time.

This work describes in detail about the method development, identification and

characterization of unknown impurities and method validation. The aim of this work was

to provide a solution for quality control laboratories for zopiclone chiral analysis.

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[4] Experimental

4.1 Chemicals and Drugs

Tablets of zopiclone and (S)-zopiclone were obtained from the local market. The

standards of racemic zopiclone, (R)- zopiclone, (S)-zopiclone (in the form of HCL salt)

were provided by Tatva Chintan Pharma Chem Pvt. Ltd., Ankleshwar, India. HPLC

grade methanol, acetonitrile, and analytical grade diethylamine (DEA), triethylamine

(TEA), trifluoroacetic acid (TFA), formic acid, acetic acid were procured from Merck.

HPLC grade water was obtained from Milli-Q water purification system.

4.2 High Performance Liquid Chromatography

The chiral separation was performed on an Agilent 1200 HPLC system consist of

a quaternary pump, column oven, photo diode array detector and an auto injector.

CHIROBIOTIC chiral columns were used for method development to separate zopiclone

enantiomers. The HPLC system was controlled and analytical data were processed using

Agilent ChemStation software (Version B.04).

4.3 Semi-preparative HPLC

Degradation impurities generated due to protic diluent, were isolated using

Agilent 1200 preparative HPLC Binary system equipped with auto sampler, diode array

detector and fraction collector. The separation was achieved on C-18 semi-prep column

using linear gradient of water and acetonitrile mobile phase. The semi-preparative HPLC

system was controlled and analytical data were processed using Agilent ChemStation

software (Version B.04).

4.4 Mass Spectroscopy

High resolution mass spectrometric data was generated using Agilent 6530

Accurate-Mass QTOF LC-MS system, connected to Agilent 1290 infinity ultra-high

performance liquid chromatography. QTOF calibrated using Agilent’s tuning mixture and

found mass error less than 1ppm. The samples were analyzed in positive polarity, MS

mode and using mobile phase consisting of 5mM ammonium formate in water and

methanol. The QTOF LC-MS system was controlled and analytical data were processed

using Agilent MassHunter software (Version B.04).

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4.5 NMR Spectroscopy

NMR experiments were performed on 400MHz Mercury plus NMR spectrometer

(Varian) in CDCl3 (containing tetramethylsilane, i.e. TMS as an internal reference

solution)) at 25°C. Proton and carbon chemical shifts were reported on δ scale in ppm,

relative to TMS (δ = 0.00ppm) as internal standard.

4.6 Chromatographic Conditions

The enantiomeric separation was optimized using a Chirobiotic TAG column

(250mm × 4.6mm, 5µm particle size, Astec) and column oven temperature maintained at

25°C. The optimal mobile phase system was consisting of methanol, trifluoroacetic acid

and diethylamine (100:0.25:0.05, v/v/v). The flow rate of the mobile phase was set at 0.3

mL/min and the analyte was detected photometrically at 304 nm. Acetonitrile used as a

diluent and the injection volume was 1 µl. The run time was set to 20 min.

4.7 Preparation of Stock Solutions

Stock solutions of racemic zopiclone, (R)-zopiclone, and (S)-zopiclone prepared

by dissolving standard samples in acetonitrile and finally filtered through a 0.45 µm pore

size Nylon filter. The stock solution concentration was fixed as 2000 µg/mL. The

working solution was also prepared in acetonitrile.

4.8 Preparation of Sample Solutions

To prepare the formulation sample, 10 tablets (2mg of (S)-zopiclone label claim)

were opened and finely ground using mortar and pestle. The ground material, equivalent

to 20 mg of (S)-zopiclone was transferred to a 10 mL volumetric flask, 7mL of

acetonitrile was added and the flask was sonicated for 10 min. Temperature of

ultrasonication bath was maintained to room temperature, i.e. 25°C. The final volume

was made up with acetonitrile and filtered through a 0.45 µm pore size Nylon filter. This

solution corresponds to analyte concentration of 2000 µg/mL further dilutions were

prepared in acetonitrile.

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4.9 Method Validation

4.9.1 Selectivity

Selectivity of the method is as its ability to measure accurately an analyte in the

presence of interferences. Selectivity of this method was indicated by the absence of any

endogenous interference at retention times of enantiomeric peaks. The absence of

interfering peak was evaluated by injecting a blank consisting of diluent and placebo.

Stability Indicating Method: The drug was subjected to forced degradation under acidic

(0.1M hydrochloric acid), basic (0.1M sodium hydroxide), and oxidative (30 % hydrogen

peroxide) stress conditions.

A. Acidic stress condition:

Acidic stress study was carried by dissolving the drug at 800 µg/mL concentration in

0.1M HCl under and kept for 30 mins in water bath at 50°C.

B. Alkaline stress condition:

An alkaline stress study was carried by dissolving the drug at 800 µg/mL concentration in

0.1M NaOH solution and kept for 30 mins in water bath at 50°C.

C. Oxidative stress condition:

The study was carried out by dissolving the drug at 800 µg/mL concentrations in 30 %

v/v hydrogen peroxide solution and kept for 30 mins in water bath at 50°C.

4.9.2 Precision

The precision of the method was checked by repeatability and intermediate

precision. Repeatability was checked by analyzing six replicate samples of (S)-zopiclone

(at analyte concentration, i.e. 2000.00 µg/mL) spiked with 0.1 % (2 µg/mL) of (R)-

zopiclone. Relative standard deviation (%RSD) of retention time and peak were

calculated for (S)- and (R)- zopiclone. The intermediate precision was determined on

different day by performing six successive injections.

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4.9.3 Linearity

Linearity corresponds to the capacity of the method to supply results directly

interval of concentration [22, 23]. Detector response linearity was assessed by preparing

twelve calibration sample solutions covering from 0.98 µg/mL to 2000 µg/mL (0.98,

1.95, 3.91, 7.81, 15.63, 31.25, 62.50, 125.00, 250.00, 500.00, 1000.00, and 2000.00

µg/mL), Regression curve was obtained by plotting peak area versus concentration, using

the least squares method.

4.9.4 Sensibility

Lower limit of detection (LLOD) defined as the lowest concentration of analyte

that can be clearly detected above the baseline signal, and was estimated at a signal to

noise ratio of 3:1. Lower limit of quantification (LLOQ) defined as the lowest

concentration of analyte that can be quantified with suitable precision and accuracy, and

was estimated at a signal to noise ration of 10:1. LLOD and LLOQ were achieved by

analyzing six injections of a series of dilute solutions, prepared for linearity study.

4.9.5 Recovery Study of (R)-zopiclone in Formulation

The standard addition and recovery experiments were conducted to determine the

accuracy of the present method. The study was carried out in triplicate by spiking placebo

with three concentrations (0.05, 0.1 and 0.15 %) of standard (R)-enantiomer and assaying

for the chromatographic method. The recovery for (R)-enantiomer was calculated from

the slope and Y-intercept of the calibration curve, drawn in the concentration range of

0.98-2000 µg/mL.

4.9.6 Ruggedness

To determine the ruggedness, the recovery experiments carried out for (R)-

enantiomer in formulation samples were again carried out in laboratory B using a

different instrument.

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4.9.7 Solution Stability

To check the solution stability of zopiclone and mobile phase, the sample was

analyzed for 24 h at room temperature, i.e., at 25°C. Resolution and composition of

zopiclone enantiomers were observed for 3, 6, 9, 12, 18, and 24 h.

4.9.8 Robustness

To evaluate the robustness of the developed method, the experimental conditions

were deliberately altered and the resolution between enantiomeric peaks was evaluated.

To study the effect of flow rate on the resolution, the flow rate changed by 10 %, i.e. 0.33

and 0.27 mL/min from the actual flow rate of 0.3 mL/min.

The effect of column temperature on resolution was studied at 22 and 28°C

instead of 25°C. The effect of the mobile phase composition was checked by varying the

composition approximately 5 % from actual value and other mobile phase components

were held constant as stated in HPLC conditions. The change in chromatographic

resolution between enantiomers was evaluated for the study.

.

Zopiclone Section-2

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[5] Result and Discussion

5.1 Method Development and Optimization

Zopiclone has no physiological charge. It has six hydrogen bond acceptors and no

hydrogen bond donor on the structure. Zopiclone has two pKa, i.e. 8.89 and 13.04, which

indicates the basic nature of the compound. The racemic sample solution of 100 µg/mL

concentration was used for the method development and optimization. To determine the

λmax, the racemic solution was scan between 200 to 400 nm using UV diode arrays

detector and we found two λmax, i.e. 220 and 304 nm (Fig. 3). Zopiclone detected at 304

nm, to achieve good detector baseline which is free from mobile phase interference.

Figure 3: UV spectra of Zopiclone

5.1.1 Selection of Chiral Stationary Phase

The objective of this study was to develop and validate short and accurate chiral

method for accurate quantification of (R) - zopiclone using macrocyclic glycopeptide

based stationary phase. In order to achieve the enantiomer separation different chiral

columns namely Chirobiotic R, Chirobiotic V, Chirobiotic T and Chirobiotic TAG were

Zopiclone Section-2

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employed. There was an indication of enantiomeric separation on Chirobiotic TAG

(Teicoplanin Aglycon) column using a mobile phase consisting of methanol.

Figure 4: Column: Chirobiotic TAG, Mobile phase: methanol, Flow: 0.75 mL/min,

Column temperature: 25°C

The CSP present in Chirobiotic TAG column is macrocyclic glycopeptide

teicoplanin aglycon covalently bonded to silica surface. The separation of zopiclone

enantiomers on this column could be due to the various interactions between the solute

enantiomers and CSP, like π- π complexation, hydrogen bonding, inclusion, dipole

stacking, and ionic binding [34].

When a stereogenic center is the part of a heterocyclic ring, such structural feature

introduces some rigidity in and around the stereogenic center and renders the two

enantiomers easily resolvable compared to stereogenic centers with four freely rotating

substituents [34]. Previous study has shown the highest enantiomeric resolution with

Chirobiotic TAG columns correspond to heterocyclic compounds, where mostly R

enantiomer eluted first than S enantiomer [35]. Zopiclone has a chiral center which is a

part of triazobicylo ring, and this may be the reason for getting the highest enantiomeric

separation on Chirobiotic TAG column.

Zopiclone Section-2

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5.1.2 Effect of Mobile Phase Modifier

To achieve symmetric peaks and base to base separation of zopiclone

enantiomers, different mobile phase combination were tried. The polar organic mode was

tried with different organic solvents, like methanol, acetonitrile, ethanol and

isopropylalcohol. The initial separation could improve, but the resulted peaks shown the

fronting (Fig. 5).

Figure 5: Mobile phase: methanol, Flow: 0.35 mL/min

In order to improve the peak shape of zopiclone enantiomers, the polar ionic

mode has introduced. The most favourable chroamatographic performance is achieved if

the iomization of solutes is controlled using optimized composition of acid and base

modifiers. The essential function of additives is to suppress ionization of strongly acidic

or basic gropus in a molecule. An alternative strategy that is sometimes effective is to

use an oppositely charged additive to promote the formation of more or less stable ion

pairs.

Zopiclone is a basic compound, and addition of acid modifier form an ionpair

that elutes through the column as an overall neutral sepcies. Different acids, like

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trifluoroacetic acid, formic acid, and acetic acid were evaluated as an acidic modifier.

During the mobile phase optimization study, the application of 0.25 % v/v TFA in to the

mobile phase addressed the peak fronting than formic acid and acetic acid (Fig. 6).

Figure 6: Mobile phase: methanol (0.25 % v/v TFA), Flow: 0.35 mL/min,

Zopiclone is a basic compound and to further improve peak shape the small

amount of diethylamine was added in to the mobile phase as a basic modifier, which may

help to block any extra active sites of stationary phase and give better peak shape (Fig. 7)

[36].

Various compositions of diethylamine in the mobile phase tried to optimize the

peak shape and enantiomeric separation, and the composition of 0.05 % gave optimum

enantiomeric separation [37]. In final method the typical retention time of both

enantiomers were about 14 and 16 min (Fig. 8).

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Figure 7: Mobile phase: methanol (0.25 % v/v TFA and 0.01 % v/v diethylamine)

Figure 8: Mobile phase: methanol (0.25 % v/v TFA and 0.05 % v/v diethylamine) Column: Chirobiotic TAG, Flow : 0.3 mL/min Column Temperature:25°C

Zopiclone Section-2

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Figure 9: Typical chiral chromatogram of (S)-zopiclone in final method

Figure 10: Typical chiral chromatogram of (S)-zopiclone sample spiked with (R)-zopiclone

Zopiclone Section-2

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In the final method, commercially available eszopiclone sample (i.e. (S)-

zopicolne) has analyzed (Fig. 9). To confirm the elution order of (R)- and (S)-

enantiomers in final method, pure (S)-enantiomer sample has spiked with undesired

enantiomer, i.e. (R)-enantiomer at 5 % w/w concentration level concentration (Fig. 10).

The undesired (R)-enantiomer was eluted first than the desired (S)-enantiomer, which

was avoided the possible interference of major (S)-enantiomer in the enantiomer purity

determination of zopiclone.

5.1.3 Influence of Diluent Solution

Initial method development was performed using methanol as a diluent solution.

During solution stability study we found that the zopiclone enantiomers underwent to the

gradual degradation and resulted into the two unknown peaks. To understand the possible

cause for zopiclone’s degradation, the solution stability was re-performed using the

diluent consisted of methanol, formic acid (100:0.05, v/v/v). The result shown sudden

increase in the compositions of respective two impurities (Fig. 11). This finding

confirmed that the degradation of zopiclone’s enantiomers is due to weak acidic nature of

methanol [37].

In developed method, the zopiclone’s degradation was proportional to the time.

During solution stability study, the zopiclone remains stable for initial 60 min of time and

degradation peaks were found after 90mins. Therefore the mobile phase composition kept

unchanged though it contains methanol & acidic modifiers because final runtime of the

method is 20 min only. After this stability study, we decided to change the diluent from

current protic solvent, i.e. methanol to improve the solution stability of zopiclone.

Time interval

(min)

% area bias Resolution

(S)-zopiclone (R)-zopiclone

Initial - - 2.09

60 0.00 0.00 2.10

90 0.21 0.18 2.08

Table 3: Zopiclone stability data in methanol diluent.

Zopiclone Section-2

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Figure 11: Solution stability of zopiclone using methanol as a diluent (a) initial profile (b) 24 h profile

Zopiclone Section-2

105

In order to overcome this challenge, the solution stability was performed in

aprotic diluent, i.e. acetonitrile and remaining all chromatographic conditions were kept

same.

Figure 12: Solution stability of zopiclone using acetonitrile as a diluent

(a) initial profile (b) 24 h profile

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The solution stability data confirmed that the zopiclone’s enantiomers remained

stable in acetonitrile diluent at room temperature for 24 h (Fig. 12). The system suitability

parameters were evaluated using methanol and acetonitrile diluent and found satisfactory

results in both the condition.

5.2 Characterization of impurities

5.2.1 Enrichment and Isolation of Impurities by Semi-Prep HPLC

Two unknown degradation products named ZPI-I and ZPI-II were eluted at

retention time of 8.9 min and 11.9 min and increased with time in the presence of

methanol. To enrich the degradation impurities, the zopiclone has forcefully degraded by

preparing the sample solution of 500 mg/10mL in methanol and kept in a water bath for

four hours at 50°C temperature.

The impurities of interest were isolated by reverse phase preparative HPLC

purification using water and acetonitrile mobile phase and XDB C-18 semi preparative

column (150x21.2mm, 5µ). The gradient conditions employed for the separation with a

linear gradient program of T (min)/%B (v/v): 0/10, 10/100, 13/100, 13.5/10, 15/10 using

a flow rate of 20 mL/min (Fig. 13). All fractions of interest were concentrated using

rotavapor at 35°C temperature.

The fractions corresponding to the unknown peaks were identified by analyzing

concentrated fractions in developed chiral HPLC method. The pure fractions

corresponding to the degradation peaks of retention time 8.9 min and 11.9 min are named

as Impurity ZPI-I and Impurity ZPI-II respectively. The structure elucidation of

impurities was performed using HRMS and NMR techniques.

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Figure 13: Preparative chromatogram of force degraded zopiclone sample

5.2.2 Structure Elucidation of Impurity ZPI-I

The characterization has started with LCMS analysis. The positive HR-MS

spectrum showed molecular ion at m/z 277.0482 (Fig. 14) confirmed the presence of

even number of nitrogen. The accurate mass data corresponded to the molecular formula

C12H9ClN4O2, which confirmed the ZPI-I had two less numbers of nitrogen than

zopiclone. The HRMS results are summarized in Table 4.

Impurity

Observed

mass of

[M+H]+

Probably molecular

formula from

observed mass

Molecular

formula of

proposed

structure

Theoretical

mass of

[M+H]+

Error

(ppm)

ZPI-I 277.0482 C12H9ClN4O2 C12H9ClN4O2 277.0487 1.74

ZPI-II 129.1022 C6H12N2O C6H12N2O 129.1022 0.23

All reported mass (amu) and molecular formulas are in [M+H] + ion form.

Table 4: HRMS data of degradation products.

Zopiclone Section-2

108

Figure 14: High resolution mass spectra of degradation product ZPI-I

NMR is the widely used and acceptable technique for the structure elucidation

and confirmation. The NMR spectra was obtained using 400 MHz instrument. Proton

NMR analysis of zopiclone and ZPI-I were recorded in CDCl3 solvent. The referencing

has done by aligning the TMS signal as 0 ppm. The residual CHCl3 and peak found at 7.3

ppm. The 1H NMR spectra of zopiclone and degradation impurity (ZPI-I) are shown in

Fig. 15 and 16 respectively.

In comparison with the zopiclone drug, the impurity ZPI-I showed the same

number of aromatic protons in their 1H NMR spectra. However, a number of protons

were found to be different in the aliphatic region. The protons corresponding to N-methyl

piperazine group were absent in ZPI-I. We found a new additional singlet with the

integration of three protons was appeared at 3.57 ppm, which confirmed the presence of

methoxy group. The singlet signal at 3.57 ppm has assigned as an integration of three

protons.

Zopiclone Section-2

109

Figure 15: 1H NMR spectra of zopiclone

Figure 16: 1H NMR spectra of degradation impurity ZPI-I

Zopiclone Section-2

110

NMR and HR-MS data confirmed the absence of piperazine group in ZPI-I.

Chemical shifts and coupling constants of Zopiclone and impurity ZPI-I are summarized

in Table 5.

Proton

Position

Zopiclone Impurity ZPI-I Impurity ZPI-II

1H

δ

(ppm) J(Hz)

1H

δ

(ppm) J(Hz)

1H

δ

(ppm) J(Hz)

Pyrazine1 d

(1H) 8.89 2.5

d (1H)

8.88 2.4

Pyrazine2 d

(1H) 8.85 2.5

d (1H)

8.82 2.4

Pyridine1 d

(1H) 8.52 9.3

d (1H)

8.39 8.8

Pyridine2 d

(1H) 8.39 2.4

d (1H)

8.46 2.5

Pyridine3 dd

(1H) 7.79

2.5, 8.8

dd (1H)

7.80 2.9, 8.8

CH s

(1H) 8.02 -

s (1H)

6.87 -

Piperazine-CH2

m (2H)

3.48-3.72

- t

(2H) 3.58 4.9

Piperazine-CH2

m (2H)

3.18-3.30

- t

(2H) 3.40 5.0

Piperazine-CH2

m (2H)

2.30-2.42

- t

(2H) 2.42 5.1

Piperazine-CH2

m (2H)

1.88-2.24

- t

(2H) 2.38 5.1

Piperazine-CH3

s (3H)

2.25 s

(3H) 2.32 -

-OMe s

(3H) 3.57 -

N-CHO s

(1H) 8.05 -

Table 5: Summary of 1H NMR shifts and coupling constants of zopiclone and its impurities

5.2.3 Structure Elucidation of Impurity ZPI-II

The 1H NMR spectra of ZPI-II did not show any aromatic protons in comparison

with zopiclone. The singlet signal at 8.02 ppm with integration value of one corresponded

to aldehyde proton of the formyl group which has attached to nitrogen atom [38]. The

Zopiclone Section-2

111

profiles of aliphatic protons were comparable with zopiclone and indicated the presence

of N-methyl piperazine ring in ZPI-II (Fig. 17).

Figure 17: 1H NMR spectra of degradation impurity ZPI-II

Figure 18: High resolution mass spectra of degradation product ZPI-II

Zopiclone Section-2

112

The positive HR-MS spectrum showed molecular ion at m/z 129.1022 (Fig. 18)

corresponding to molecular formula C6H12N2O. The probable reason for the formation of

these impurities could be the degradation at carbamate bond (R-O(C=O)N-R,R) of

zopiclone. The characterization data confirmed that the ZPI-I is 4-methylpiperazine-1-

carbaldehyde. This further confirmed by matching the retention time with reagent grade

4-methylpiperazine-1-carbaldehyde (CAS registry number, 7556-55-0).

We could not found the exact literature precedence, which explains the possible

mechanism for formation of ZPI-I and ZPI-II from zopiclone. The HRMS and NMR data

support the below proposed structures of the degradation impurities (Fig. 19).

Figure 19: Proposed chemical structure of zopiclone impurities

Zopiclone Section-2

113

5.3 Results of Method Validation

5.3.1 Results of System suitability

The system suitability results were evaluated using methanol and acetonitrile

as a diluent and results are summarized in Table 6.

Diluent TR TS NR NS Rs α

Acetonitrile 1.05 1.17 10132 10033 2.01 1.13

Methanol 1.14 1.23 10430 10231 2.30 1.15

(TR: USP tailing factor for R isomer; TS: USP tailing factor for S isomer; NR: number of theoretical plates for R isomer, NS: number of theoretical plates for S isomer; Rs: USP resolution, α: enantioselectivity)

Table 6: System suitability results.

Peak purity of both the enantiomers was passing using a diode array detector.

Report of peak purity is presented in Fig. 20 and 21.

Figure 20: Peak purity report of (R)-zopiclone

Zopiclone Section-2

114

Figure 21: Peak purity report of (S)-zopiclone

5.3.2 Results of Selectivity

To evaluate the selectivity, the chromatogram obtained by analyzing blank run

consisting of diluent and placebo was compared in order to check the absence of any

peaks likely to interfere at RTs of (S)- and (R)- zopiclone. As it can be seen in overlay of

zopiclone (LLOQ level) and blank chromatogram (Fig. 15), blank chromatograms are

free from any interference at RTs of zopiclone enantiomers. The peak purity factor was

within the calculated threshold limit for (R)-zopiclone and (S)-zopiclone enantiomers

(Table 7).

Zopiclone Purity Factor Threshold

(R)-enantiomer 999.331 999.093

(S)-enantiomer 999.523 999.011

Table 7: Peak purity results.

Zopiclone Section-2

115

Figure 22: Overlay of blank and racemic zopiclone chromatograms

Stability Indicating Method

The degradation products were separated from zopiclone enantiomers hence the

developed method was found to be stability indicating and results are free from any

Stress conditions % Degradation Peak purity result

Acid degradation (0.1M HCL) 18 Pass

Alkali degradation (0.1M NaOH) 07 Pass

Oxidative degradation (30 % H2O2) 01 Pass

Table 8: Peak purity results for force degradation study.

Zopiclone Section-2

116

interference. The purity factor is within the threshold limit for zopiclone enantiomers in

forced degradation samples (Table 8).

5.3.3 Results of Method Precision

Precision Data

Sr. No. (R)-zopiclone (S)-zopiclone

RT Area RT Area

1 14.41 975 15.926 967

2 14.4 977 15.906 972

3 14.4 993.2 15.906 989.2

4 14.41 990.4 15.925 984.2

5 14.43 987 15.93 981.8

6 14.41 987.2 15.92 982.2

Average 14.4 985 15.9 979.4

SD 0.01 7.34 0.01 8.25

% RSD 0.05 0.75 0.07 0.84

Intermediate Precision Data

(R)-zopiclone (S)-zopiclone

RT Area RT Area

1 14.83 932 15.53 967

2 14.46 911 15.91 972

3 14.93 902 15.91 989

4 14.53 908 15.93 984

5 14.44 922 15.93 982

6 14.63 917 15.92 982

Average 14.6 915.3 15.9 979.3

SD 0.2 10.73 0.16 8.19

% RSD 1.38 1.17 1.01 0.84

Table 9: Results of precision study

Zopiclone Section-2

117

Repeatability was checked by analyzing six replicate racemic samples of

Zopiclone. Relative standard deviation (% RSD) of retention time and area under the

peaks were calculated for (S)- and (R)- zopiclone.

The intermediate precision was determined in another laboratory by performing

six successive injections. In intermediate precision study, results showed that % RSD

values were in the same order of magnitude than those obtained for repeatability. The

results for method precision and robustness are summarized in Table 9.

5.3.4 Results of linearity

The results show that good correlation existed between the peak area and

concentration of undesired (R)-enantiomer. The described method was linear over the

wide concentration range from 0.98 to 2000 µg/mL. The regression was found to be

linear all over the wide concentration range and correlation coefficients values were

1.0000 and 0.9999 for (R)- and (S)- zopiclone respectively (Fig. 23 and 24).

Concentration

(ppm) Area

0.95 3.7

1.90 6.7

3.81 13.3

7.62 27.7

15.23 57.4

30.47 118.6

60.94 242.1

121.88 474.3

243.75 971

487.50 1945.3

975.00 3939.1

1950.00 7979.5

Figure 24 : Linearity results for (R)-zopiclone

1

10

100

1000

10000

Pea

k A

rea

(R)-zopiclone

Y = 4.09 X - 12.45

r2= 1.0000

Concentration (ppm)

Zopiclone Section-2

118

Concentration

(ppm) Area

0.95 3.9

1.90 7

3.81 12.9

7.62 27.3

15.23 58.2

30.47 119.7

60.94 239.3

121.88 471.5

243.75 971.1

487.50 1940.5

975.00 3878.5

1950.00 7954.7

Figure 25 : Linearity results for (S)-zopiclone

5.3.5 Results of Sensitibility

The results corresponding to LLOD and LLOQ study were found satisfactory. The

concentration for LLOQ and LLOD were estimated to be 1950 and 980 ng/mL for both

enantiomers respectively. The results are summarized in Table 10.

(R)-zopiclone (S)-zopiclone

LOD (ng/mL) 980 980

S/N 4.1 4.6

LOQ (ng/mL) 1950 1950

S/N 12.5 10.8

Table 10: Results of Sensibility

1

10

100

1000

10000

Pea

k A

rea

(S)-zopiclone

Y = 4.06 X - 13.48

r2= 0,9999

Concentration (ppm)

Zopiclone Section-2

119

5.3.6 Results of (R)-zopiclone Recovery Study in Formulation

The recovery experiments were conducted to determine the accuracy of the

present method for the quantification of (R)-zopiclone in formulation samples. (R)-

zopiclone was spiked to the extracted (S)-zopiclone sample in triplicate at 0.05, 0.1 and

0.15% of target analyte concentration (i.e. 2000 µg/mL). Recovery was calculated from

the slope and Y-intercept of the calibration curve obtained in linearity study. The same

recovery experiments were also conducted using a different system in laboratory B at the

same concentration levels as tested in laboratory A. The results were well in agreement.

This confirmed the ruggedness of the method and the results of recovery study are

summarized in Table 11.

% Level of test

concentration

Added

(ng) Recovered (ng) % Recovery

%

RSD

Laboratory

A

80 1600 1523 95.2 6.2

100 2000 1940 97 3.9

120 2400 2352 98 3.1

Laboratory

B

80 1600 1505 94 5.5

100 2000 1910 95.5 5.1

120 2400 2410 98 4.8

Table 11: Recovery result of (R)-zopiclone in enteric coated formulation

5.3.7 Results of Solution Stability

No significant change was observed in resolution and peak area composition of

zopiclone enantiomers during the solution stability study. The data are presented in Table

12, It can be seen from the data that % bias of area for zopiclone enantiomers was less

than 1 % hence sample solution and mobile phase are stable for 24h at room temperature,

i.e., at 25°C.

Zopiclone Section-2

120

Time interval (h)

% area bias

Resolution

(S)-zopiclone

(R)-zopiclone

Initial - - 2.02

3 0.13 0.12 2.02

6 -0.15 -0.19 2.01

9 -0.18 -0.22 2.06

12 -0.14 -0.17 2.04

18 -0.12 -0.13 2.03

24 -0.19 0.21 2.01

Table 12: Results of solution stability study

5.3.8 Results of Robustness

In robustness study, the racemic zopiclone sample was analyzed with change of

different experimental conditions as a part of robustness study. The resolution between

(R)- and (S)- zopiclone peaks were remain more than 1.75 for all deliberately changed

chromatographic conditions and this confirmed the robustness of the method. The results

are summarized in Table 13.

Zopiclone Section-2

121

Parameters Resolution between two enantiomers

Flow rate (mL/min)

0.27 2.21

0.3 2.00

0.33 1.96

Column temperature (°C)

22 2.11

25 2.06

28 2.01

TFA content (%, v/v)

0.24 1.81

0.25 2.04

0.26 1.90

DEA content (%, v/v)

0.04 1.77

0.05 1.99

0.06 2.10

Table 13: Results of robustness study

Zopiclone Section-2

122

[6] Conclusion

The stability indicating chiral HPLC method was described for the enantiomeric

separation of zopiclone in pharmaceutical formulation.

The baseline separation was achieved on a Chiralcel TAG column, is made by

covalently bonding the aglycon part of teicoplanin to silica gel via linkage chains. The

percentages of acidic and basic additives in the mobile phase have shown significantly

influence to the peak shape and resolution of the zopiclone enantiomers. The baseline

separation was achieved with a run time of 20min using mobile phase consisted of

methanol, trifluoroacetic acid and diethylamine (100:0.25:0.05, v/v/v).

This is the first report to describe the validated chiral HPLC method for the

enantioselective analysis of zopiclone in pharmaceutical formulation using macrocyclic

glycopeptide stationary phase. In this study we found the importance of an aprotic diluent

to achieve solution stability of zopiclone up to 24h at room temperature. We observed

two unknown degradation impurities, which has been enriched, isolated and characterized

by NMR and HRMS techniques. The most probable structures were proposed for these

impurities based on the spectral data. The method was validated showing satisfactory

data for all the tested validation parameters and the method was found to be sensitive and

linear over the thousand fold concentration range. This method can be used for routine

analysis in quality control laboratories.

Zopiclone Section-2

123

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