81

CHAPTER-III

CABERGOLINE

82

3.1. INTRODUCTION

Cabergoline is a dopamine receptor agonist D2 receptors. In vitro, rat

studies show cabergoline has a direct inhibitory effect

on pituitary lactotroph (prolactin) cells[1]. It is frequently used as a first-line

agent in the management of prolactinomas due to higher affinity for D2

receptor sites, less severe side effects, and more convenient dosing schedule

than the older bromocriptine. Although cabergoline is commonly described

principally as a dopamine D2 receptor agonist, it also possesses significant

affinity for the D3, D4,5-HT1A, 5-HT2A, 5-HT2B, 5-HT2C, α2B- receptors, and

moderate/low affinity for the D1 and 5-HT7receptors. Cabergoline functions as

an agonist at all receptors except for 5-HT7 and α2B-, where it acts as an

antagonist[2].

Figure: 3.A. Structure of Cabergoline

IUPAC Name : (1-[(6-allylergolin8β-yl)- carbonyl]-1-[3-

(dimethylamino)propyl]-3-ethylurea

Formula : C26H37N5O2

Molecular Weight : 451.6

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Routes : Oral

It is used to treat different types of medical problems that occur when

too much of the hormone prolactin is produced. Cabergoline was invented by

Farmitalia-Carlo Erba SpA in Milan in 1981/82[3]. The drug was approved by

the FDA on December 23, 1996. It went generic in late 2005 following US

patent expiration. It can be used to treat certain menstrual problems, fertility

problems in men and women, and pituitary prolactinomas[4] (tumors of the

pituitary gland). Cabergoline is also sometimes used to treat Parkinson's

disease [4,5] (a disorder of the nervous system that causes difficulties with

movement, muscle control, and balance). It works by stopping the brain from

making and releasing the prolactin hormone from the pituitary gland.

Cabergoline use is usually stopped when prolactin levels are normal for 6

months. It may be given again if symptoms of too much prolactin occur again.

It has at times been used as an adjunct to SSRI antidepressants. It also

has been suggested online that it has a possible recreational use in reducing

or eliminating the male refractory period[7]. It is also used by bodybuilders to

control gynecomastia caused by elevated prolactin levels through the use of

anabolic steroids such as nandrolone and trenbolone. Additionally, a

systematic review and meta-analysis concluded that prophylactic treatment

with cabergoline reduces the incidence, but not the severity, of ovarian

hyperstimulation syndrome (OHSS), without compromising pregnancy

outcomes, in females undergoing stimulated cycles of in vitro fertilization

(IVF)[7]. Also, a study on rats found that cabergoline reduces voluntary alcohol

consumption, possibly by increasing GDNF expression in the ventral

tegmental area[8].

84

Side effects are mostly dose dependent. Much more severe side effects

are reported for treatment of Parkinson's disease and (off-label treatment)

for restless leg syndrome which both typically require very high doses. The

side effects are considered mild when used for treatment of

hyperprolactinemia and other endocrine disorders or gynecologic indications

where the typical dose is 10-100 times smaller than for Parkinson's disease.

Along with pergolide in causing valvular heart disease.[11][12], so FDA removed

pergolide from the U.S. market on March 29, 2007[13]. Since cabergoline is not

approved in the U.S. for Parkinson's Disease, but for hyperprolactinemia, the

drug remains on the market[14][15].

3.2. LITERATURE SURVEY

E. Pianezzola et al [14] developed the Determination of cabergoline in plasma

and urine by high-performance liquid chromatography with electrochemical

detection. A sensitive and selective high-performance liquid chromatographic

method for the determination of cabergoline in plasma and urine has been

developed. After buffering plasma and urine samples, cabergoline was

extracted with a methylene chloride—isooctane mixture, back-extracted into

0.1 M phosphoric acid, then analysed by reversed-phase high-performance

liquid chromatography. Quantitation was achieved by electrochemical

detection of the eluate. The linearity, precision and accuracy of the method

were evaluated. No interference from the biological matrices (human plasma

and urine) was observed. The assay was still inadequate in terms of sensitivity

for the quantitation of cabergoline plasma concentrations after a single oral

dose of 1 mg of the drug to humans, but was successfully used in the

determination of the urinary excretion of the drug.

85

A.Onal et al [15] developed the Selective LC determination of cabergoline in

the Bulk Drug and in tablets: In Vitro Dissolution Studies.In this cabergoline

(CAB) is an ergot alkaloid derivative with dopamine agonist activity. A novel,

simple, and rapid stability-indicating high-performance liquid

chromatographic (HPLC) method for assay of CAB in tablets has been

developed and validated. Chromatography was performed on a 4.6 mm

i.d. × 250 mm, 5 μm particle, cyano column with acetonitrile–10 mM

phosphoric acid, 35:65 (v/v), containing 0.04% triethylamine, as mobile phase,

at a flow rate of 1.0 mL min−1, and UV detection at 280 nm. Response was a

linear function of concentration in the range 0.1–4 μg mL−1(r 2 = 0.9999). The

recovery of the method was good (99.45%) and RSD values for intra-day and

inter-day precision were 0.24–0.88% and 0.66–1.19%, respectively. The

method can be used for quality-control assay of CAB in tablets, for stability

studies, and for in vitro dissolution studies.

3.3. EXPERIMENTAL

3.3.1. Instrumentation

To develop a liquid chromatographic method for quantitative

estimation of using an isocratic peak HPLC instrument on a Chromosil C18

column (250 mm x 4.6 mm, 5μ). The instrument is isocratic pump equipped

with PEAK LC-UV7000 variable wavelength detector. A 20μL Hamilton syringe

was used for injecting the samples. Data was analyzed by using Peak

Chromatographic software version 1.06. A Denwar balance was used for

weighing the materials.

86

3.3.2. Chemicals and Solvents

The reference sample of cabergoline (API) was obtained from V.V.MED

Pharmaceuticals, Hyderabad. The formulation was procured from the local

market. Acetonitrile, water used were of HPLC grade and purchased from

Merck Specialities Private Limited, Mumbai, India. Potassium dihydrogen

phosphate and ortho phosphoric acid were AR grade purchased from local

market.

3.3.3. The buffer solution

About 0.1N sodium dihydrogenphosphate was prepared with water

and filtered through 0.45μ nylon filter.

3.3.4. The mobile phase

A mixture of acetonitrile, 1% ortho phosphoric acid and sodium

dihydrogen phosphate (40:30:30)

3.3.5. Standard solution of the drug

10mg of cabergoline was taken in a 10ml volumetric flask and 10ml of

mobile phase was added to obtain 1000µg/ml of cabergoline. 0.5 ml of the

stock solution was pipetted out into a 100ml volumetric flask and made up to

the mark. The resulting solution was filtered through nylon filter paper. The

calibration curve was plotted with the five concentrations of 5.0-25µg/ml of

working standard solutions. Calibration solutions were prepared daily and

analyzed immediately after preparation.

3.4. METHOD DEVELOPMENT

For developing the method, 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

87

appropriate wave length and choice of stationary and mobile phases. The

following studies were conducted for this purpose.

3.4.1. Detection wavelength

The spectrum of diluted solutions of the cabergoline in methanol was

recorded separately on UV spectrophotometer. The peak of maximum

absorbance wavelength was observed. The spectra of the both cabergoline

were showed that a wavelength was found to be 275 nm.

3.4.2. 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 (250 mm x 4.6 mm, 5μm) column.

3.4.3. Selection of the mobile phase

In order to get sharp peak and base line separation of the components,

the author has 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

acetonitrile, 1% ortho phosphoric acid and sodium dihydrogen phosphate

(40:30:30) 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.

88

3.4.4. Flow rate

Flow rates of the mobile phase were changed from 0.5–1.2 mL/min for

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

the 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.

3.4.5. Optimized chromatographic conditions

Chromatographic conditions as optimized above were shown in Table 3.1.

These optimized conditions were followed for the determination of

cabergoline in bulk samples and its combined tablet formulations. The

chromatograms of standard and sample were shown in Figure 3.B, 3.C, 3.D.

89

Table 3.1. Optimized method conditions for cabergoline

Mobile phase Acetonitrile, 1% ortho phosphoric acid and

sodium dihydrogenphosphate (40:30:30)

Pump mode Isocratic

Mobile phase PH 2.9

Diluents Mobile phase

Column chromosil C18 column (250 mm x 4.6 mm, 5μ)

Column Temp Ambient

Wavelength 275 nm

Injection Volume 20 μl

Flow rate 1.0 mL/min

Run time 5min

Retention Time 1.655min

Mobile phase PH 4.2

90

Fig: 3.B: Blank Chromatogram for Cabergoline

Fig: 3.C: Standard Chromatogram for Cabergoline

Fig: 3.D; Typical RP-HPLC Chromatogram for Cabergoline

91

3.5. VALIDATION OF THE PROPOSED METHOD

The proposed method 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.

3.5.1. System suitability

System suitability of the method was evaluated by analyzing the repeatability,

peaks symmetry (Symmetry factor), theoretical plates of the column, peak

area and retention time. System suitability tests were carried out on freshly

prepared standard stock solutions of cabergoline. System suitability results

of cabergoline were shown in Table 3.2

Table: 3.2. System suitability results

System suitability Parameter Result

Retention time 1.65min

Area 157841

Theoretical plates 2290

Tailing factor 1.17

3.5.2. 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. Specificity results of cabergoline

were shown in Table 3.3.

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Table 3.3 Specificity results

Test Retention time (minutes)

Standard 1.65

Formulation 1.60

Linearity 1.62

Precision 1.64

3.5.3. Linearity

Linearity was performed by preparing mixed standard solutions of cabergoline

at different concentration levels including working concentration mentioned

in experimental condition i.e.5µg/ml. Twenty micro liters of each

concentration was injected in duplicate into the HPLC system. The response

was read at 275 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 Table 3.4.

3.5.4. Precision

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

operational conditions. Precision of the method was performed as Intra-day

precision, Inter day precision.

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3.5.4.A. Intra-day precision

To study the Intra-day precision, six replicate standard solution (15µg/ml) of

cabergoline was injected. The percent relative standard deviation (% RSD) was

calculated and it was found to be 0.946, which

are well within the acceptable criteria of not more than 2.0. Results of system

precision studies are shown in Table 3.5.

Table 3.4: Linearity results

Level

Concentration of

cabergolineIn µg/ml

Mean peak area

Level -1 5µg/ml 75313.1

Level -2 10µg/ml 139545.0

Level -3 15µg/ml 212414.4

Level -4 20µg/ml 275380.4

Level -5 25µg/ml 345005.9

Range: 5µg/ml to

25µg/ml

Slope

Intercept

Correlation coefficient

13504.42

6965.46

0.999

94

On X axis concentration of sample, On Y axis peak area response

Figure: 3.D. Calibration curve

Table 3.5. Intra-day precision results

SAMPLE

CONC

(µG/ML)

INJECTION

NO PEAK AREA

R.S.D

(Acceptance

criteria ≤2.0%)

Cabergoline

15

1 209319.8

0.946

2 207619.9

3 205102.8

4 208721.3

5 208297.9

6 204610.1

3.5.4.B. Inter Day precision

To study the interday precision, six replicate standard solution

(15µg/ml) of cabergoline was injected on third day of sample preparation. The

percent relative standard deviation (% RSD) was calculated and it was found

0

50000

100000

150000

200000

250000

300000

350000

400000

0 5 10 15 20 25 30

a

r

e

a

conc. in ppm

cabergoline

95

to be 0.892 which was well within the acceptable criteria of not more than

2.0. Results of system precision studies are shown in Table 3.6.

Table .3.6. Inter Day precision

Sample

Conc.

(in

µg/ml)

Injection No. Peak Areas

RSD

(Acceptance

criteria ≤

2.0%)

Cabergoline 15

1 214743.0

0.892

2 212895.0

3 210053.8

4 211177.0

5 210983.4

6 214202.7

3.5.5. 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 50%, 100% and 150% 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

3.7 Satisfactory recoveries ranging from 99.66 to 100.16 were obtained by the

proposed method. This indicates that the proposed method was accurate.

96

Table: 3.7. Accuracy results

Level

Target (In µg/ml)

Amount of Pantoprazole spiked (µg/ml)

Total (µg/ml)

Amount of Pantoprazole recovered(µg/ml)

% Recovery

%RSD

50 %

10 5 15 15.27 101.08 0.64

10 5 15 14.97 99.8

10 5 15 15.05 100.3

100%

10 10 20 20.01 100.05 0.63 10 10 20 20.10 100.5

10 10 20 19.85 99.25

150%

10 15 25 25.14 100.56 0.12 10 15 25 25.10 100.4

10 15 25 25.08 100.32

Mean % of recovery 100.25

Mean RSD =0.46

3.5.6. Ruggedness

Ruggedness test was determined between two different analysts, instruments

and Columns. The value of RSD was below 2.0%, showed ruggedness of

developed analytical method. The results were presented in Table 3.8.

Table: 3.8: Ruggedness results

Sample

Conc.

(in µg/ml)

Injection

No.

Peak

Areas

RSD

(Acceptance

criteria ≤ 2.0%)

Cabergoline 15 1 213458

0.6 2 212693

97

3 214108

4 214419

5 216589

6 215577

3.5.7. 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.

Cabergoline at 5 µg/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.

3.5.8. Limit of detection and Limit of quantification

To determine the Limit of Detection the sample was dissolved by using

Mobile Phase and injected until peak was diapered. After 0.4µg/ml dilution,

Peak was not clearly observed. So it confirms that 0.4µg/ml is limit of

Detection and Limit of Quantification was found to be 1.5µg/ml. For this study

six replicates of the analyte at lowest concentration were measured and

quantified. The LOD and LOQ of cabergoline are given in Table 3.10

Table: 3.9: Robustness results

S.NO Parameter Change Area % Of

Change

1 Standard ……….. 212414 …………

2 Mobile

phase

Acetonitrile, 1% OP and

sodium

211408 0.48

98

dihydrogenphosphate

(35:30:35)

3 Acetonitrile, 1% OP and

sodium

dihydrogenphosphate

(45:30:25)

212449 0.1

4 Wavelength 273nm 210356 0.97

5 277nm 212009 0.11

6 Mobile

phase PH

4.0 213098 0.32

7 4.5 212299 0.06

Table: 3.10: LOD and LOQ results

Parameter Measured volume

Limit of Quantification 1.5µg/ml

Limit of Detection 0.4µg/ml

3.5.9. Formulation:

For assay Dostinex 20 tablets were weighed and calculate the average

weight. Accurately weighed and transfered the sample equivalent to 10mg of

cabergoline in to a 10ml volumetric flask. Diluent was added and sonicate to

dissolve it completely and make volume up to the mark with diluents. Mix

well and filter through 0.45um filter. Further pipette 1ml of the above stock

solution into a 10ml volumetric flask and dilute up to mark with diluents and

finally 20µg/ml were prepared. Mix well and filter through 0.45um filter. An

aliquot of this solution was injected into HPLC system. Peak area of

cabergoline was measured for the determination. The percentage of

cabergoline in tablet was found to be 99.9%

99

3.6. DISCUSSION ON THE RESULTS

RP-HPLC method developed for the determination of drugs has great

importance in the quality control analysis. The chromatograms for pure drug

were obtained by using different mobile phases like methanol, acetonitrile,

THF and different buffers like sodium dihydrogen phosphate in different

volume ratios. Different columns like C8, C18, phenyl, cyano with different

dimension were used. Then retention time and tailing factor were calculated.

Finally acetonitrile, 1% ortho phosphoric acid and sodium dihydrogen

phosphate (40:30:30) and C18 analyzed column were selected which gave a

sharp and symmetrical peak with 1.17 tailing factor.

Calibration graph was found to be linear in the range 5.0-25µg/ml. Five

different concentrations of cabergoline in the given range were prepared and

injected into HPLC. The slope (m) and intercept(c) obtained were found to be

13504.42 and 6965.46 respectively. A plot drawn between peak area and

concentration of drug solution range studied was found to have excellent

linear correlation with a correlation coefficient of 0.999. The LOQ and LOD of

cabergoline were found to be 1.5µg/ml and 0.4µg/ml respectively indicating

the sensitivity of the method.

The difference between theoretical added amount and practically

achieved amount is called accuracy of analytical method. Accuracy was

determined at 3 different level 50%, 100% and 150% of the target

concentration in duplicate. Result of accuracy data presented in Table 3.7. The

percentage assay of cabergoline in formulation was found between 99.8-

101.08 indicates the good recovery. Method repeatability/intermediate

precision was assessed from the assay of six samples by two different analysts

using different chromatographic systems on different days. The

100

chromatographic results are summerised on Table 3, the % RSD was found

less than 2.0 %, and hence the repeatability is well within the acceptance

criteria. The results were shown in Table 3.5 and 3.6. This method has been

applied to investigate the samples of cabergoline in marketed brands and the

percentage of assay is in good agreement with the labeled

The method involves significantly reduced runtime with better peak

shape. The method found to be linear, accurate, rugged and robust for

validated parameters. This method offers better turnaround of analytical

values. Using the same method, assay was performed for individual samples

and found that values were in good agreements. Hence this method is

considered to be an excellent method. for the assay determination and

content uniformity of cabergoline sodium in oral solid dosage form.

3.7. REFERENCES

1. "Dostinex at www.rxlist.com". Retrieved 2007-04-27.

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aqueous humor dynamic modulation in the Cynomolgus monkey eyes".

Experimental Eye Research 88 (3): 386–97..

3. US Patent 4526892 - Dimethylaminoalkyl-3-(ergoline-8'.beta.carbonyl)-

ureas

4. Sayyah-Melli, M; Tehrani-Gadim, S; Dastranj-Tabrizi, A; Gatrehsamani,

F; Morteza, G; Ouladesahebmadarek, E; Farzadi, L; Kazemi-Shishvan, M

(2009). "Comparison of the effect of gonadotropin-releasing hormone

agonist and dopamine receptor agonist on uterine myoma growth.

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journal 30 (8): 1024–33.

5. Sankaran, S.; Manyonda, I. (2008). "Medical management of fibroids".

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6. Krüger TH, Haake P, Haverkamp J, et al. (December 2003). "Effects of

acute prolactin manipulation on sexual drive and function in males".

Journal of Endocrinology 179 (3): 357–65.

7. Youssef MA, van Wely M, Hassan MA, et al. (March 2010). "Can

dopamine agonists reduce the incidence and severity of OHSS in

IVF/ICSI treatment cycles? A systematic review and meta-analysis".

Hum Reprod Update 16 (5): 459–66.

8. Carnicella, S.; Ahmadiantehrani, S.; He, D. Y.; Nielsen, C. K.; Bartlett, S.

E.; Janak, P. H.; Ron, D. (2009). "Cabergoline Decreases Alcohol

Drinking and Seeking Behaviors Via Glial Cell Line-Derived Neurotrophic

Factor". Biological Psychiatry 66 (2): 146–153.

9. Schade, Rene; Andersohn, Frank; Suissa, Samy; Haverkamp, Wilhelm;

Garbe, Edeltraut (2007-01-04). "Dopamine Agonists and the Risk of

Cardiac-Valve Regurgitation". New England Journal of Medicine 356

(1): 29–38.

10. Zanettini, Renzo; Antonini, Angelo; Gatto, Gemma; Gentile, Rosa; Tesei,

Silvana; Pezzoli, Gianna (2007-01-04). "Valvular Heart Disease and the

Use of Dopamine Agonists for Parkinson's Disease". New England

Journal of Medicine 356 (1): 39–46.

11. "Food and Drug Administration Public Health Advisory". 2007-03-29.

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102

12. Low dose cabergoline for hyperprolactinaemia is not associated with

clinically significant valvular heart disease

13. Treatment with low doses of cabergoline is not associated with

increased prevalence of cardiac valve regurgitation in patients with

hyperprolactinaemia

14. E. Pianezzole, V. Belloti, R. La Corix and M. Strolin Benedetti,

Determination of cabergoline in plasma with electrochemical

detection, Journal of chromatography B: Biomedical sciences and

Applications, Volume 574, Issue 1, 7 February 1992, pages 170-174.

15. A. Onal, O. Sagirh and D. sensoy, Selective LC Determination of

Cabergoline in the Bulk Drug and in Tablets: In Vitro Dissolution

Studies, Chromatographia, volume 65, pages 561-567.