CHAPTER 2

ZOPICLONE

55

2.1. DRUG PROFILE

Zopiclone is a central nervous system depressant and belongs to non

benzodiazepine sedative and hypnotic. It is used to treat insomnia where sleep

initiation or sleep maintenance are prominent symptoms. Long term use is not

recommended as tolerance, dependence, addiction can occur with prolonged use.[1-2]

.

Zopiclone is a cyclopyrrone compound that has been reported to possess

hypnotic, muscle relaxant, and anticonvulsant properties analogous to benzodiazepine

compounds such as diazepam. Chemically [3]

it is 4-methyl-1piperazine-carboxylic

acid- 6-(5-chloro-2-pyridinyl)-6, 7dihydro-7-oxo-5H-pyrrolo[3,4-b]pyrazin-5-yl ester.

Zopiclone belongs to the group of medicines called central nervous system (CNS)

depressants. The therapeutic pharmacological properties of Zopiclone

include hypnotic, anxiolytic, anticonvulsant, and myorelaxant properties [4]

.

Zopiclone is known colloquially as a "Z-drug". Zopiclone, as traditionally sold

worldwide, is a racemic mixture of two stereoisomers, only one of which is active.[5-6]

It is recommended that Zopiclone be taken on an "as needed" basis. Daily or

continuous use of the drug is not usually advised.[7]

Zopiclone is a tranquillizer drug.

It works by causing a depression or tranquillisation of the Central Nervous System.

As Zopiclone is sedating it is marketed as a sleeping pill.

Figure 2.A: Structure of Zopiclone

56

Systematic (IUPAC) name : (RS)-6-(5-chloropyridin-2-yl)-7-oxo-6,7-

dihydro-5H-pyrrolo[3,4-b]pyrazin-5-yl 4-

methylpiperazine-1-carboxylate

Formula : C17H17ClN6O3

Molecular mass : 388.808 g/mol

Bioavailability : 52-59% bound to plasma protein

Metabolism : Various cytochrome P450 liver enzymes

Excretion : Urine

Routes : Oral tablets.

List of brand names of Zopiclone:

S.No.

BRAND

NAME

FORMULATION AVAILABLE

STRENGTH

MANUFACTURER

1 LYZOP TABLET 7.5mg Hetero

2 ZOLINOX TABLET 7.5mg Ranbaxy

3 ZOLIUM TABLET 7.5mg FDC

Table 2.1

Zopiclone, a benzodiazepine-like drug was introduced and initially promoted

as having less dependence and withdrawal than traditional benzodiazepine drugs.

However, Zopiclone may have an even greater addictive potential than

benzodiazepines and has been described as a "benzodiazepine in disguise".[8-10]

Tolerance to the effects of Zopiclone can develop after a few weeks. Long term use

should be avoided. Abrupt withdrawals particularly with prolonged and high doses

can in severe cases cause seizures and delirium.[11-12]

Publications in the British Medical Journal do not give any evidence to the

claim that Zopiclone has a low dependence potential. In fact, physical dependence and

recreational abuse and withdrawal syndromes similar to those seen in benzodiazepine

withdrawal are frequently encountered. Withdrawal symptoms included anxiety,

tachycardia, tremor, sweats, flushes, palpitations, derealisation, and further

57

insomnia.[13]

Suspected withdrawal convulsions during detoxification from Zopiclone

has been reported, however the individual was a high dose Zopiclone misuser.[14]

The risk of dependency on Zopiclone when used for less than 2 weeks or only

used occasionally is low.[15]

However, this is disputed by one study of low dose

Zopiclone taken for only 7 nights. It found that discontinuation of

Zopiclone caused significant rebound insomnia. Furthermore when midazolam taken

for 7 nights was discontinued no rebound insomnia occurred suggesting that

Zopiclone may have even more significant problems of tolerance and dependence

than the benzodiazepines.[16]

After 3 weeks of use mild to moderate rebound

withdrawal symptoms appear upon discontinuation of Zopiclone.[17]

Due to the risk of

tolerance and physical dependence, Zopiclone is only recommended for short term

(1–4 weeks max) relief of insomnia, or alternatively, long term infrequent use.[18]

Long-term Zopiclone users who have become physically dependent should not

discontinue their medication abruptly as severe withdrawal symptoms may occur such

as delirium.[19]

If Zopiclone has been taken for more than a few weeks then the

medication should be gradually reduced or preferably to cross over to an equivalent

dose of diazepam (Valium), which has a much longer half life which makes

withdrawal easier and then gradually taper their dosage over a period of several

months in order to avoid extremely severe and unpleasant withdrawal symptoms (e.g.,

inner restlessness, psychomotor agitation, abdominal pain, hypertension,

hallucinations, seizures, anxiety, depression, psychosis, etc.) which can last up to two

years after withdraw if the withdrawal is done too abruptly.[20-22]

After 4 weeks of nightly use of Zopiclone day time withdrawal related anxiety

begin to emerge in some users. However, the day time withdrawal anxiety does not

appear to be as intense as that seen with the much shorter acting triazolam which

provokes even more profound day time withdrawal anxiety symptoms in long term

users.[23]

According to the World Health Organization, Zopiclone, although molecularly

is not a benzodiazepine, binds unselectively with high affinity to the same

benzodiazepine sites that the benzodiazepine class of drugs do. The World Health

58

Organization also stated that Zopiclone is cross tolerant with benzodiazepines and one

can substitute one for the other. The withdrawal symptoms from Zopiclone reported

included anxiety, tachycardia, tremor, sweating, rebound insomnia, derealisation,

convulsions, palpitations and flushes.[24]

Zopiclone is cross tolerant with benzodiazepines.[25]

Alcohol has cross

tolerance with GABAA receptor positive modulators such as the benzodiazepines and

the nonbenzodiazepine drugs. For this reason alcoholics or recovering alcoholics may

be at increased risk of physical dependency on Zopiclone.

To withdraw from Zopiclone sleeping tablets cross over to an equivalent dose

of diazepam, the equivalency table by Professor Heather Ashton can be used.[26]

To

withdraw and reduce the intensity of withdrawal symptoms, including interdose

withdrawal symptoms, it is important that the blood levels of a drug remain constant

throughout a 24 hour period. This is not possible with Zopiclone as it is a short acting

drug; therefore users of Zopiclone should cross over to an equivalent dose of

diazepam to begin their dose taper.[27]

While it acts on the same benzodiazepine

receptors as the benzodiazepine family of drugs it is not classed as a benzodiazepine

(with which it shares a number of characteristics and effects) due to its differing

molecular structure. Zopiclone is classed as a cyclopyrrolone derivative.[28]

59

2.2. LITERATURE SURVEY

Several analytical methods have been reported for the determination of

Zopiclopne in pure drug, pharmaceutical dosage forms and in biological samples

using spcetrophotometry, liquid chromatography, electro kinetic chromatography high

performance thin layer chromatography either in single or in combined forms.

Beata Paw and Genowefa Misztal et al

[29] were developed a rapid, simple

and accurate chromatographic (HPLC) and spectrophotometric methods for the

determination of Zopiclone in tablets were elaborated. Acetonitrile was found to be a

suitable extraction solvent. The samples were chromatographed on Nova-Pak C18

column and UV detection at 304 nm. The elution was achieved isocratically with a

mobile phase of 0.067 M phosphate buffer pH 7.95 — acetonitrile (55:45, v/v).

Diazepam was applied as an internal standard. The method was validated for

precision, linearity, accuracy and limit of detection. The recovery (mean±SD) in

HPLC was 99.85±0.04% and in the UV-spectrophotometry 100.08±0.09%.

Hiren N

et al[30]

were developed a A simple, selective and sensitive

isocratic HPLC method with triple quadrupole mass spectrometry detection has been

developed and validated for simultaneous quantification of Zopiclone and its

metabolites in human plasma. The analytes were extracted using solid phase

extraction, separated on Symmetry shield RP8 column (150 mm × 4.6 mm i.d.,

3.5 μm particle size) and detected by tandem mass spectrometry with a turbo ion

spray interface. Metaxalone was used as an internal standard. The method had a

chromatographic run time of 4.5 min and linear calibration curves over the

concentration range of 0.5–150 ng/mL for both Zopiclone and N-desmethyl Zopiclone

and 1–150 ng/mL for Zopiclone-N-oxide. The intra-batch and inter-batch accuracy

and precision evaluated at lower limit of quantification and quality control levels were

within 89.5–109.1% and 3.0–14.7%, respectively, for all the analytes. The recoveries

calculated for the analytes and internal standard were ≥90% from spiked plasma

samples. The validated method was successfully employed for a comparative

bioavailability study after oral administration of 7.5 mg Zopiclone (test and reference)

to 16 healthy volunteers under fasted condition.

60

J. Van Bocxlaer et al [31]

were developed a new HPLC method for Zopiclone

(Imovane) in postmortem specimens by GC-MS and HPLC with diode-array

detection. Blood or stomach contents (1 ml) were mixed with 50 µl N-

allylnormetazocine (internal standard; 0.4 mg/ml in methanol) and thereafter with 1

ml ethanol, 1M-K2CO3 (5 drops, pH 9) and 2 ml H2O. The mixture was extracted

with 8 ml methyl tert-butyl ether/n-hexane (3:1). After mixing for 15 min, samples

were centrifuged for 10 min at 2500 g. The organic layer was evaporated at room

temperature under N2 and redissolved in 100 µl mobile phase. A 50 µl portion was

analyzed by HPLC on a 5 µm Hypersil BDS C18 column (15 cm × 4.6 mm i.d.),

1.5M-ammonium acetate/acetonitrile/ methanol/H2O (solvent A, 3:10:10:77; solvent

B, 3:40:40:17) as mobile phase with gradient elution from 5-50% B within 20 min (1

ml/min) and diode-array detection. Calibration graphs were linear from 0.9-10 µg/ml

Zopiclone and 2-40 µg/ml pentazocine. GC-MS analysis of liver samples was also

performed on a column (30 m × 0.25 mm i.d.) coated with DB-5 (0.25 µm) operated

with temperature programming from 150-270°C at 30°C/min and then to 290°C (held

for 5 min) at 1°C/min, He as carrier gas (0.8 ml/min) and mass-selective ion-trap

detection. Details of a liquid-liquid extraction procedure for extraction of the drugs

from the liver are presented. Without the use of these procedures, Zopiclone can be

easily overlooked during routine forensic screening

Gottfried Blaschke et al

[32] were report the preparative separation of

rac-Zopiclone using malic acid as the resolving agent. Furthermore, two different

methods for the analytical determination of Zopiclone enantiomers by HPLC on chiral

stationary phases are described. The benzodiazepine receptor binding of the isolated

enantiomers was investigated. Half-maximal inhibitory concentrations of (+)- and (−)-

Zopiclone were 21 or 1,130 nmol/liter, respectively, indicating a more than 50 times

higher affinity of the (+)-enantiomer toward the receptor.

Lakshmana Rao et al [33]

were developed an A simple, rapid, sensitive

and precise HPLC method has been developed for the estimation of Zopiclone in pure

and tablet dosage form. In this method RP-C18 column (100mmx4.6mm I.D., 3µm

particle size) with mobile phase consisting of 0.02M phosphate buffer and acetonitrile

in the ratio of 55:45v/v in a isocratic mode was used. The detection wavelength is

61

304nm and the flow rate is 1ml/min. The linearity found in the range of 20-100µg/ml

and shows a correlation coefficient of 0.9913. The proposed method was validated by

determining sensitivity, linearity, accuracy and precision. This method is simple, fast,

accurate, precise and reproducible hence can be applied for the routine quality control

analysis of Zopiclone in pure and pharmaceutical dosage form.

Frank T.Peters et al [34]

were developed a Reliable analytical data are a

prerequisite for correct interpretation of toxicological findings in the evaluation of

scientific studies, as well as in daily routine work. Unreliable analytical data might

not only be contested in court, but could also lead to unjustified legal consequences

for the defendant or to wrong treatment of the patient. This is especially true in the

context of quality management and accreditation, which have become matters of

increasing relevance in analytical toxicology in recent years. In this paper, the author

tried in this chapter an important considerations in analytical method validation will

be discussed which may be used as guidance by scientists wishing to develop and

validate analytical methods. Therefore, new analytical methods to be used in forensic

and/or clinical toxicology require careful method development and thorough

validation of the final method.

62

2.3. EXPERIMENTAL

2.3.1. Instrumentation

Peak HPLC containing LC 20 AT pump, variable wavelength programmable

uv/vis detector and Rheodyne injector was employed for the investigation. The

chromatographic analysis was performed on a Chromosil C18 column (250 mm x 4.6

mm, 5μm) column. Degassing of the mobile phase was done by using a Loba

ultrasonic bath sonicator. A Denver balance was used for weighing the materials.

2.3.2. Chemicals and Solvents

The reference sample of Zopiclone (API) was obtained from Lupin,

Ahmadabad. The Formulation was procured from the local market. Acetonitrile,

Methanol, Tetra hydro furan (THF) and orthophosphoric acid used were of HPLC

grade and purchased from Merck Specialties’ Private Limited, Mumbai, India.

2.3.3. The buffer solution

About 1.0 mL of orthophosphoric acid was diluted to 1000 mL with water.

This solution was mixed and pH was adjusted to 5.8 with ortho phosphoric acid and

filtered through 0.45μ nylon filter.

2.3.4. The mobile phase

A mixture of Acetonitril: Methanol: THF: 0.1%OPA in the ratio of 45:20:5:30

v/v was prepared and used as mobile phase.

2.3.5. Standard solution of the drug

For analysis we 100 ppm standard solution was prepared in mobile phase.

Required concentrations were obtained from 100 ppm standard solution by proper

dilution.

2.3.6. Sample (tablet) solution

The formulation tablets of Zopiclone (ZOLINOX – 7.5mg) were crushed to

give finely powdered material. With Powder we prepared 70 ppm solution in mobile

phase and then filtered through Ultipor N66 Nylon 6, 6 membrane sample filter paper.

63

2.4. METHOD DEVELOPMENT

For developing the method [35-43]

, a systematic study of the effect of various

factors was undertaken by varying one parameter at a time and keeping all other

conditions constant. Method development consists of selecting the appropriate wave

length and choice of stationary and mobile phases. The following studies were

conducted for this purpose.

2.4.1. Detection wavelength

The spectrum of diluted solutions of the Zopiclone in mobile phase was

recorded separately on UV spectrophotometer. The peak of maximum absorbance

wavelength was observed. The spectra of the both Zopiclone were showed that a

wavelength was found to be 303 nm.

2.4.2. Choice of stationary phase

Preliminary development trials have performed with octadecyl columns with

different types, configurations and from different manufacturers. Finally the expected

separation and shapes of peak was succeeded Chromosil C18 column.

2.4.3. Selection of the mobile phase

In order to get sharp peak, low tailing factor and base line separation of the

components, we carried out a number of experiments by varying the composition of

various solvents and its flow rate. To effect ideal separation of the drug under

isocratic conditions, mixtures of solvents like methanol, water and Acetonitrile with

or without different buffers indifferent combinations were tested as mobile phases on

a C18 stationary phase. A mixture of Acetonitril: Methanol: THF : 0.1%OPA in the

ratio of 45:20:5:30 v/v was proved to be the most suitable of all the combinations

since the chromatographic peak obtained was better defined and resolved and almost

free from tailing.

2.4.4. Flow rate

Flow rates of the mobile phase were changed from 0.5 – 1.5 mL/min for

optimum separation. A minimum flow rate as well as minimum run time gives the

64

maximum saving on the usage of solvents. It was found from the experiments that 1.0

mL/min flow rate was ideal for the successful elution of the analyte.

2.4.5. Optimized chromatographic conditions

Chromatographic conditions as optimized above were shown in Table 2.2

These optimized conditions were followed for the determination of Zopiclone in bulk

samples and its combined tablet Formulations. The chromatograms of standard and

sample were shown in Figure 2.B

Mobile phase ACN45% ,MEOH ,20% ,THF 5% ,

0.1 %OPA 30%

Pump mode Isocratic

Mobile phase PH 4.5

Diluent The mobile phase

Column chromosil C18 column (250 mm x 4.6

mm, 5μ)

Column Temp Ambient

Wavelength 303 nm

Injection Volume 20 μl

Flow rate 1.0 mL/min

Run time 10 min

Retention Time 3.3 min

Table 2.2: Optimized chromatographic conditions for estimation Zopiclone

65

Figure 2.B: RP - HPLC Chromatogram of standard solution

66

2.5. VALIDATION OF THE PROPOSED METHOD

The proposed method [44-53]

was validated as per ICH guidelines. The

parameters studied for validation were specificity, linearity, precision, accuracy,

robustness, system suitability, limit of detection, limit of quantification, and solution

stability.

2.5.1. Specificity

The specificity of method was performed by comparing the chromatograms of

blank, standard and sample. It was found that there is no interference due to excipients

in the tablet formulation and also found good correlation between the retention times

of standard and sample. The specificity results are shown in Table 2.3

NAME OF THE SOLUTION Retention Time in Minutes

BLANK NO PEAKS

Zopiclone 3.03

Table 2.3: Specificity study

2.5.2 Linearity

Linearity was performed by preparing mixed standard solutions of Zoplicone

at different concentration levels including working concentration mentioned in

experimental condition i.e. 0.8µg/ml. Twenty micro liters of each concentration was

injected in duplicate into the HPLC system. The response was read at 303 nm and the

corresponding chromatograms were recorded. From these chromatograms, the mean

peak areas were calculated and linearity plots of concentration over the mean peak

areas were constructed individually. The regressions of the plots were computed by

least square regression method. Linearity results were presented in Table2.4

67

Figure 2.C: On X axis concentration of sample solution, On Y axis peak area

response Linearity results of Zopiclone:

Level Concentration of OLMESARTAN in µg/ml Mean peak area

Level -1 0.2 76001.1

Level -2 0.4 142422.3

Level -3 0.6 219516.2

Level -4 0.8 295425.0

Level -5 1.0 373769.3

Level -6 1.2 465371.4

Level-7 1.4 5381693.1

Range:

0.2 to 1.4

µg/ml

Slope

Intercept

Correlation coefficient

390754.9

0.029

0.9993

Table 2.4: Linearity Results

2.5.3. Precision

Precision is the degree of repeatability of an analytical method under normal

Operational conditions. Precision of the method was performed as intraday precision,

Inter day precision.

-100000

0

100000

200000

300000

400000

500000

600000

0 2 4 6 8

68

2.5.3.1: Intraday precision

To study the intraday precision, five replicate standard solution of Zopiclone

was injected. The percent relative standard deviation (% RSD) was calculated and it

was found to be 1.697, which are well within the acceptable criteria of not more than

2.0. Results of system precision studies are shown in Table 2.5.

SAMPLE

CONC(µg/ml) INJECTION No. PEAKS

AREA

R.S.D

(Acceptance

criteria 2.0)

Zopiclone

0.8

1 279249.2

1.697

2 277905.2

3 268011.3

4 273292.3

5 271282.8

Table 2.5: System Suitability (Intra Day)

2.5.5. Inter Day precision

To study the inter day precision, five replicate standard solution of Zopiclone

was injected on third day of sample preparation. The percent relative standard

deviation (% RSD) was calculated and it was found to be 1.743, which are well within

the acceptable criteria of not more than 2.0. Results of system precision studies are

shown in Table 2.6.

69

SAMPLE

CONC(µg/ml) INJECTION No. PEAKS

AREA

R.S.D

(Acceptance

criteria 2.0)

Zopiclone

0.8

1 290196.4

1.743

2 295425.0

3 295018.1

4 303377.6

5 291464.5

Table 2.6: System Precision (Inter Day)

2.5.6. Accuracy

The accuracy of the method was determined by standard addition method. A

known amount of standard drug was added to the fixed amount of pre-analyzed tablet

solution. Percent recovery was calculated by comparing the area before and after the

addition of the standard drug. The standard addition method was performed at 20%,

40% and 60% level. The solutions were analyzed in triplicate at each level as per the

proposed method. The percent recovery and % RSD was calculated and results are

presented in Table 2.7 Satisfactory recoveries ranging from 99.9 to 100.1 were

obtained by the proposed method. This indicates that the proposed method was

accurate.

70

LEVEL

Amount of

OLMESARTAN

spiked (µg/ml)

Amount of

OLMESARTAN

recovered(µg/ml)

%

Recovery

MEAN %

Recovery

%R.S.D

MEAN

%

R.S.D

20 %

1.2 1.19 99.16

0.484

1.2 1.18 98.33

1.2 1.19 99.16

40%

1.4 1.38 98.57

0.422

1.4 1.37 97.85

1.4 1.38 98.57

60%

1.6 1.56 97.5

1.099

1.6 1.56 97.5

1.6 1.59 99.37

98.44 0.668

Table 2.7: Percentage Recovery and %RSD

71

2.5.7. Robustness

The robustness study was performed by slight modification in flow rate of the

mobile phase, pH of the buffer and composition of the mobile phase. Zopiclone at

0.4µg/ml concentration was analyzed under these changed experimental conditions. It

was observed that there were no marked changes in chromatograms, which

demonstrated that the developed method was robust in nature. The results of

robustness study are shown in Table 2.8.

Condition Mean area % assay % difference

Unaltered 142422.3 100.0 0.0

Flow rate at 0.8 mL/min

Flow rate at 1.2mL/min

142874.5

141966.7

100.3

99.68

0.3

0.32

Mobile phase:

MeOH: water

82% 18%

78% 22%

142588.2

143120.6

100.11

100.49

0.11

0.49

pH of mobile phase at 4.8 143058.1 100.44 0.44

pH of mobile phase at 4.2 142688.0 100.1 0.1

Table 2.8: Robustness

72

2.5.8. System suitability

System suitability was studied under each validation parameters by injecting

six replicates of the standard solution. The system suitability parameters are given in

Table 2.9.

Parameter Tailing factor Theoretical plates

Specificity study 1.30 5557.18

Linearity study 1.45 4227.53

Precision study 0.99 2555.7

Table 2.9: System Suitability

2.5.9. Limit of detection and Limit of quantification

Limit of detection (LOD) is defined as the lowest concentration of analyte that

gives a detectable response. Limit of quantification (LOQ) is defined as the lowest

Concentration that can be quantified reliably with a specified level of accuracy and

Precision. For this study six replicates of the analyte at lowest concentration were

Measured and quantified. The LOD and LOQ of Zopiclone given in Table 2.10

Parameter Measured volume

Limit of Quantification 45ng/ml

Limit of Detection 10ng/ml

Table 2.10: LOQ and LOD

73

2.6. RESULTS AND DISCUSSION

To optimize the RP-HPLC parameters, several mobile phase compositions

were tried. A satisfactory separation and good peak symmetry was found in a mixture

of Acetonitril: Methanol: THF: 0.1%OPA in the ratio of 45:20:5:30 v/v and 1.0

mL/min flow rate proved to be better than the other mixtures in terms of resolution

and peak shape. The optimum wavelength for detection was set at 303nm at which

much better detector responses for drug was obtained. As it was shown in Fig. 2.B the

retention times were 3.30 min for Zopiclone. The number of theoretical plates was

found to be 5557.8, which indicates efficient performance of the column. A system

suitability test was applied to representative chromatograms for various parameters.

The results obtained were within acceptable limits and are represented in Table 2.9.

Thus, the system meets suitable criteria.

The calibration curve was obtained for a series of concentration in the range

of 0.2-1.4µg/ml and it was found to be linear. Seven points graphs was constructed

covering a concentration range 0.2-1.4µg/ml. The standard deviation of the slope and

intercept were low. The data of regression analysis of the calibration curves are shown

in Table 2.C.

The proposed method has been applied to the assay of T commercial tablets

containing Zopiclone. Sample was analyzed for five times after extracting the drug as

mentioned in assay sample preparation of the experimental section. The results

presented good agreement with the labeled content. Low values of standard deviation

denoted very good repeatability of the measurement. Thus it was showing that the

equipment used for the study was correctly and hence the developed analytical

method is highly repetitive. For the intermediate precision a study carried out by the

same author working on the same day on two consecutive days indicated a RSD of

1.697 & 1.743. This indicates good method precision.

The system suitability parameter like capacity factor, asymmetry factor, tailing

factor and number of theoretical plates were also calculated. It was observed that all

the values are within the limits. The statistical evaluation of the proposed method was

revealed its good linearity, reproducibility and its validation for different parameters

and let us to the conclusion that it could be used for the rapid and reliable

determination of Zopiclone in tablet formulation.

74

All these factors lead to the conclusion that the proposed method is accurate,

precise, simple, sensitive and rapid and can be applied successfully for the estimation

of Zopiclone in bulk and in pharmaceutical formulations without interference and

with good sensitivity

75

2.7. BIBILOGRAPHY

1. "What's wrong with prescribing hypnotics?‖; Drug Ther Bull; 2004, 42 (12):

89–93.

2. Touitou Y; "Sleep disorders and hypnotic agents: medical, social and

economical impact" (in French); Ann Pharm Fr; 2007; 65 (4): 230–8.

3. Budavari S; The Merck Index, 13th

Edn, Merck & Co., Inc., Whitehouse

Station, NJ. 2004; 10247.

4. Jonathan U; The Australian Drug Guide. Melbourne: Bookman Press Pvt.

Ltd.; 2000; p. 743.

5. Blaschke, G; Hempel, G; Müller We; "Preparative and analytical separation

of the Zopiclone enantiomers and determination of their affinity to the

benzodiazepine receptor binding site.‖; Chirality; 1993; 5(6): 419–21.

6. Fernandez, C; Maradeix, V; Gimenez, F; Thuillier, A; Farinotti, R;

"Pharmacokinetics of Zopiclone and its enantiomers in Caucasian young

healthy volunteers.‖; Drug metabolism and disposition: the biological fate of

chemicals 1993; 21(6): 1125–8.

7. Van, Der, Kleijn, E; "Effects of Zopiclone and temazepam on sleep, behaviour

and mood during the day"; European journal of clinical pharmacology; 1989;

36(3): 247–51.

8. Bramness JG, Olsen H; "Adverse effects of Zopiclone". Tidsskrift for den

Norske laegeforening; 1998; 118 (13): 2029–32.

9. Luty S, Sellman D; "Imovane—a benzodiazepine in disguise". N. Z. Med. J;

1993; 106 (959): 293.

10. Deveaux M, Chèze M, Pépin G; "The role of liquid chromatography-tandem

mass spectrometry (LC-MS/MS) to test blood and urine samples for the

toxicological investigation of drug-facilitated crimes". Ther Drug Monit,

2008; 30 (2): 225–8.

11. "Hypnotic dependence: zolpidem and Zopiclone too". Prescrire Int; 2001;

10 (51): 15.

12. Wong CP, Chiu PK, Chu LW. "Zopiclone withdrawal: an unusual cause of

delirium in the elderly". Age Ageing; 2005; 34(5): 526–7.

76

13. Ones IR, Sullivan G; "Physical dependence on Zopiclone: case reports"; BMJ;

1998; 316 (7125): 117.

14. Aranko, K; Henriksson, M; Hublin, C; Seppäläinen, Am; ―Misuse of

Zopiclone and convulsions during withdrawal"; Pharmacopsychiatry; 1991;

24 (4): 138–40.

15. Anderson, Aa; "Zopiclone and nitrazepam: a multicenter placebo controlled

comparative study of efficacy and tolerance in insomniac patients in general

practice‖; 1987; 10(1): 54–62.

16. Begg, Ej; Robson, Ra; Frampton, Cm; Campbell, Je; ―A comparison of

efficacy and tolerance of the short acting sedatives midazolam and

Zopiclone"; The New Zealand medical journal; 1992; 105 (944): 428–9.

17. Dorian, P; Sellers, Em; Kaplan, H; Hamilton, C; "Evaluation of Zopiclone

physical dependence liability in normal volunteers"; Pharmacology; 1983;

27(2): 228–34.

18. Mendelson WB, Jain B; "An assessment of short-acting hypnotics"; Drug Saf;

1995; 13(4): 257–70.

19. Harter C, Piffl-Boniolo E, Rave-Schwank M; "Development of drug

withdrawal delirium after dependence on zolpidem and zoplicone" (in

German); Psychiatr Prax; 1999; 26 (6): 309.

20. sanofi-aventis Canada Inc ;"IMOVANE (Zopiclone) Tablets, 5.0 mg and 7.5

mg".

21. Kahlert I; Brüne M; "A case of primary Zopiclone dependence"; Dtsch Med

Wochenschr; 2008; 126 (22): 653–4.

22. Heather Ashton; "Benzodiazepines: how they work and how to withdraw‖.

23. Fontaine, R; Beaudry, P; Le, Morvan, P; Beauclair, L; Chouinard, G;

"Zopiclone and triazolam in insomnia associated with generalized anxiety

disorder: a placebo-controlled evaluation of efficacy and daytime anxiety";

International clinical psychopharmacology; 1990; 5 (3): 173–83.

24. WHO (2006); "World Health Organisation - Assessment of Zopiclone";

who.int.http://www.who.int/medicines/areas/quality_safety/4.6ZopicloneCritR

eview.pdf.

77

25. Cohen, C; Sanger, Dj; ―Tolerance, cross-tolerance and dependence measured

by operant responding in rats treated with triazolam via osmotic pumps‖;

Psychopharmacology; 1994; 115 (1-2): 86–94.

26. EQUIVALENCY TABLE.

27. Explanation by Dr JG McConnell in his article called The

Clinicopharmacotherapeutics of Benzodiazepine and Z drug dose Tapering

Using Diazepam.

28. Elie, R; Deschenes, Jp; "Efficacy and tolerance of Zopiclone in insomniac

geriatric patients"; International pharmacopsychiatry; 1982; 17(2): 179–87.

29. ―Determination of Zopiclone in tablets by HPLC and UV-spectrophotometry‖;

Journal of Pharmaceutical and Biomedical Analysis; 2000; 23(5): 819-823.

30. ―Validated method for simultaneous quantification of Zopiclone and its

metabolites, N-desmethyl Zopiclone and Zopiclone-N-oxide in human

plasma‖; Journal of Chromatography B; 2008; 864(1-2,15): 137-148.

31. J Anal Toxicol; ―Analysis of Zopiclone (Imovane) in postmortem specimens

by GC-MS and HPLC with diode-array detection‖; RSC Journals.; 1996;

20(1):52-4.

32. ―Preparative and analytical separation of the Zopiclone enantiomers and

determination of their affinity to the benzodiazepine receptor binding site‖;

Chirality; 1993; 5(6), 419–421.

33. ―RP-HPLC Method for the Estimation of Zopiclone in Tablet Dosage Form‖;

International Journal of Pharmaceutical Research; 2011; 3(1): 49-51.

34. Forensic Science International; 2007; 165(2): 216-224.

35. ―Q2A: Text on Validation of Analytical Procedures‖; International Conference

on Harmonization; Federal Register; 1995; 60(40): 11260–11262.

36. ―Q2B: Validation of Analytical Procedures: Methodology; Availability‖;

International Conference on Harmonization; Federal Register; 1997; 62(96):

27463–27467.

37. "Analytical Procedures and Methods Validation: Chemistry, Manufacturing

and Controls Documentation; Availability"; FDA; Federal Register (Notices);

2000; 65(169): 52776 – 52777.

38. www.fda.gov/cder/guidance/cmc3.pdf.

78

39. USP 25–NF 20; ―Validation of Compendial Methods Section (1225) (United

States Pharmacopeal Convention, Rockville, Maryland, USA, 2002)‖; 2256.

40. G.A. Shabir; ―Validation of HPLC Chromatography Methods for

Pharmaceutical Analysis‖; Understanding the Differences and Similarities

between Validation Requirements of FDA, the US Pharmacopeia and the ICH;

J. Chromatogr. A; 2003; 987(1-2): 57-66.

41. C.E. Wood; "Medicare Program; Changes to the Hospital Outpatient

Prospective"; Med. J. Aust; 1996; 165: 510–514.

42. A. Prentice; "Medical Management of Menorrhagia": Br. Med. J; 1999; 319,

1343–1345.

43. D.T. Baired and A.F. Glasier; "Hormonal Contraception"; New Engl. J. Med;

1993; 328: 1543–1549.

44. P.E. Belchetz; "Hormonal Treatment of Postmenopausal Women"; New Engl.

J. Med; 1994; 330: 1062–1071.

45. International Conference on Harmonization (ICH) of Technical Requirements

for the Registration of Pharmaceuticals for Human Use; ―Validation of

analytical procedures: definitions and terminology‖; Geneva (1996).

46. U.S. FDA; Title 21 of the U.S. Code of Federal Regulations:

21 CFR 211—Current good manufacturing practice for finished

pharmaceuticals.

47. U.S. FDA - Guidance for Industry (draft) Analytical Procedures and Methods

Validation: Chemistry, Manufacturing, and Controls and Documentation,

2000.

48. ISO/IEC 17025, General requirements for the competence of testing and

calibration laboratories, 2005.

49. International Conference on Harmonization (ICH) of Technical Requirements

for the Registration of Pharmaceuticals for Human Use, Validation of

analytical procedures: Methodology, adopted in 1996, Geneva.

50. U.S. EPA, Guidance for methods development and methods validation for the

Resource Conservation and Recovery Act (RCRA) Program, Washington,

D.C. (1995).http://www.epa.gov/sw-846/pdfs/methdev.pdf.

79

51. General Chapter 1225, Validation of compendial methods, United States

Pharmacopeia 30, National Formulary 25, Rockville, Md., USA, The United

States Pharmacopeial Convention, Inc., (2007).

52. U.S. FDA - Guidance for Industry, Bioanalytical Method Validation.

53. G. C. Hokanson, A life cycle approach to the validation of analytical methods

during pharmaceutical product development, Part I: The initial validation

process, Pharm. Tech., Sept. 1994, pp. 118–130.