54

CHAPTER-II

ANASTROZOLE

55

2.1 INTRODUCTION

Anastrozole is an aromatase-inhibiting drug approved for treatment

of breast cancer after surgery, as well as for metastasis in both pre and post-

menopausal women. This medication is used in women, who have

experienced menopause, as a first treatment of breast cancer that has spread

within the breast or to other areas of the body. This medication is also used to

treat breast cancer in women whose breast cancer has worsened after taking

tamoxifen (Nolvadex).

Fig: 2.A Structure of Anastrozole

Systematic (IUPAC) name : 2, 2’-[5-(1H-1, 2, 4-triazol-1-ylmethyl)-

1,3 phenylene]bis(2-methylpropanenitrile)

Formula : C17H19N5

Mol. mass : 293.4

Routes : Oral

Excretion : 11% renal

The studies suggested that anastrozole is the preferred medical

therapy for postmenopausal women with localized breast cancer that is

56

estrogen receptor (ER) positive[1]. Another study found that the risk of

recurrence was reduced to 40%, which also included an increased risk of bone

fractures, and those ER negative patients benefitted from switching to

anastrozole[2]. The severity of breast cancer is increased by estrogen, as sex

hormones cause hyperplasia, and differentiation at estrogen receptor sites[3].

Anastrozole works by inhibiting the synthesis of estrogen.

Anastrozole binds reversibly to the aromatase enzyme through

competitive inhibition, inhibits the conversion of androgens to estrogens in

peripheral tissues (outside the CNS), and a few CNS sites in various regions

within the brain[4][5]. Anastrozole has been tested for reducing estrogens,

including estradiol, in men[6]. Excess estradiol in men can cause benign

prostatic hyperplasia, gynecomastia, and symptoms of hypogonadism.

Serious adverse reactions with anastrozole occurring in less than 1 in

10,000 patients, are: 1) skin reactions such as lesions, ulcers, or blisters 2)

allergic reactions with swelling of the face, lips, tongue, and/or throat. This

may cause difficulty in swallowing and/or breathing, 3) changes in blood tests

of the liver function, including inflammation of the liver with symptoms that

may include a general feeling of not being well, with or without jaundice, liver

pain or liver swelling.

Common adverse reactions (occurring with an incidence of > 10%) in

women taking anastrozole included: hot flashes, asthenia, arthritis, pain,

arthralgia, pharyngitis, hypertension, depression, nausea and vomiting, rash,

osteoporosis, fractures, back pain, insomnia, pain, headache, bone pain,

peripheral edema, increased cough, dyspnea, pharyngitis and lymphedema.

57

2.2 LITERATURE SURVEY

Mendes et al [7] described a rapid, sensitive and specific method for

quantifying the aromatase inhibitor (anastrozole) in human plasma using

dexchlor pheniramine as the internal standard (I.S.). The analyte and the I.S.

were extracted from 200 µl of human plasma by liquid-liquid extraction using

a mixture of diethyl ether: dichloromethane (70:30, v/v) solution. Extracts

were removed and dried in the organic phase then reconstituted with 200 µl

of acetonitrile: water (50:50; v/v). The extracts were analyzed by high

performance liquid chromatography coupled with photospray tandem mass

spectrometry (HPLC-MS-MS). Chromatography was performed isocratically on

a Genesis, C18 4 microm analytical column (100 mm x 2.1mm i.d.). The

method had a chromatographic run time of 2.5 min and a linear calibration

curve ranging from 0.05-10 ng ml. The limit of quantification (LOQ) was 0.05

ng ml. This HPLC-MS-MS procedure was used to assess pharmacokinetic

studies.

Sathis Kumar et al [8] developed and validated a simple, precise and

accurate method by using a simple solvent system for anastrozole bulk and

tablet dosage form. In the developed method, water and ethanol are used as

solvents. The λmax was determined to be 221nm. The procedure was

validated as per ICH rules for Accuracy, Precision, Detection limit, Linearity,

Reproducibility and Quantitation limit. The linearity concentration range was

40-60µg/mL with the correlation coefficient of 0.9971. The percentage

recovery for anastrozole was found to be 98.6 to 100.8%. Limit of detection

and limit of quantitation values were found to be 1µg/mL and 3µg/mL. The

58

method has been successfully used to analyze commercial solid dosage

containing 1mg of anastrozole with good recoveries and proved to be robust.

D. Sathis Kumar et al [9] described a simple, economic, accurate

reverse phase isocratic HPLC method for the quantitation of anastrozole in

tablet dosage form. The quantification was carried out using Gracesmart

RP18, 5 μ (100 mmx4.6 mm) with UV detected at 215 nm. The elution was

achieved isocratically with a mobile phase comprising a mixture of buffer (pH

6.0) and acetonitrile (1:1, v/v). The flow rate was 1.0 mL/min The procedure

was validated as per ICH rules for Accuracy, Precision, Detection limit,

Linearity, Reproducibility and Quantitation limit. The linearity concentration

range was 10-20 mcg/mL with the correlation coefficient of 0.9935. The

percentage recovery for anastrozole was found to be 97.31±2.2%. Limit of

detection and limit of quantitation values were found to be 0.351µl/ml and

1.053µl/ml. The method has been successfully used to analyze commercial

solid dosage containing 1mg of anastrozole with good recoveries and proved

to be robust.

LIANG Meng et al [10] determined anastrozole in Compound Feed Usin

g HPLC. Aanastrozole was extracted from compound feed by diethyl ether and

dichloromethane. The extract was centrifuged and the supernatant was purifi

ed by MCX-SPE, and then tested by HPLC. Quantitative analysis was carried ou

t by UV-detector and qualitative analysis was by DAD. The result showed that

the concentration of anastrozole and peak area was linear correlation betwee

n 0.1~1.000 μg/ml, r=0.9989. The reclaim rate was 90%~112% with cofficient

of variation less than 5%. The LDL of anastrozole was 1 μg/ml. Four medicines

have no disturbance to anastrozole test under this condition.

59

HIRIYANNA, S. G et al [11] detected three impurities ranging from

0.08%-0.12% by peak area in anastrozole active pharmaceutical ingredient

detected by simple isocratic reverse-phase high performance liquid

chromatography (HPLC). These impurities were isolated by prep-HPLC and

were characterized by LC-MS/MS, GCMS and NMR experimental data. Based

on the results obtained from different spectroscopic experiments, these

impurities have been characterized as 2,2’-(5-((4H-1,2,4-triazol-4-yl)methyl)-

1,3-phenylene)bis(2-methylpropanenitrile) (Impurity I), 2,2’-(5-methyl-1,3-

phenylene)bis(2-methylpropanenitrile) (Impurity II) and 2,2’-(5-

(bromomethyl)-1,3-phenylene)bis(2-methyl-propanenitrile) (Impurity III).

2.3. EXPERIMENTAL

2.3.1. Instrumentation

Chromatographic separation was performed on a PEAK

chromatographic system equipped with LC-P7000 isocratic pump; Rheodyne

injector with 20μl fixed volume loop, variable wavelength programmable UV

detector UV7000 and the output signal was monitored and integrated by

PEAK. Chromatographic Software version 1.06. ElicoSL 159 UV-2301 double

beam UV-Visible spectrophotometer was used to carry out spectral analysis

and the data was recorded by Hitachi software. Sonicator (Loba-1.5L).

Ultrasonicator was used to sonicating the mobile phase and samples.

Standard and sample drugs were weighed by using Denver electronic

analytical balance (SI-234) and pH of the mobile phase was adjusted by using

Systronics digital pH meter.

60

2.3.2. Chemicals and Solvents

The reference sample of anastrozole (API) was obtained from SMS

Pharma, Hyderabad. The Formulation was procured from the local market.

Acetonitrile, methanol, water used were of HPLC grade and purchased from

Merck Specialties Private Limited, Mumbai, India.

2.3.3. The mobile phase

A mixture of methanol, water and acetonitrile (50:30:20) v/v was

prepared and used as mobile phase.

2.3.4. Standard solution of the drug

For analysis 100µg/ml standard solution was prepared. Required

concentrations were obtained from 100µg/ml solution by proper dilution.

2.3.5. Sample (tablet) solution

The formulation tablets of anastrozole (Redest -1mg) were crushed to

give finely powdered material. Using the powdered material of the drug, a

solution of 10µg/ml is prepared by dissolving appropriate quantity of the drug

material and then filtered through Ultipor N66 Nylon (6,6) membrane sample

filter paper.

2.4. 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

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

following studies were conducted for this purpose.

61

2.4.1. Detection wavelength

The spectra of diluted solutions of the anastrozole in methanol were

recorded separately on UV spectrophotometer. The peak of maximum

absorbance wavelength was observed. The spectra of the both anastrozole

were showed that a wavelength was found to be 218 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 with intersil C18 column (250 mm x 4.6 mm, 5μ).

2.4.3. Flow rate

Flow rates of the mobile phase were changed from 0.6 – 1.5 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.

2.4.4. Selection of the mobile phase

In order to get sharp peaks and base line separation of the

components, number of experiments were conducted by varying the

commonly used solvents, their compositions, flow rates and wavelength. A

variety of mobile phases were investigated in the development of an HPLC

method suitable for analysis of anastrozole. 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

62

tested as mobile phases and columns are used. The few conditions used when

optimization during method development were given below.

Table2.1 Trial: 1 Chromatographic condition

S.No Parameter Conditions

1 Mobile phase Methanol:water 80:20 v/v

2 Column C18

3 Wavelength 218nm

4 Flow rate 1ml/min

5 Runtime 8min

Fig: 2.B Chromatogram of Trial

Result: Because of merged peaks it was not selected as system suitable peak

and further trials were conducted.

63

Table: 2.2 Trial: 2 Chromatographic conditions

S.No Parameter Conditions

1 Mobile phase Methanol:ACN 80:20 v/v

2 Column C18

3 Wavelength 218nm

4 Flow rate 1ml/min

5 Runtime 5min

Fig: 2.C Chromatogram of Trial

Result: Because of high tailing and low theoretical plates it was not selected

as system suitable peak and further trials were conducted.

Table: 2.3 Trial: 3 Chromatographic conditions

64

S.No Parameter Conditions

1 Mobile phase Acetonitrile:water 80:20

v/v

2 Column C18

3 Wavelength 218nm

4 Flow rate 1ml/min

5 Runtime 8min

Fig: 2.D Chromatogram of Trial

Result: Because of low theoretical plates it was not selected as system

suitable peak and further trials were conducted.

65

Table: 2.4 Trial: 4 Chromatographic conditions

S.No Parameter Conditions

1 Mobile phase Methanol:water:ACN (80:10:10 v/v)

2 Column C18

3 Wavelength 218nm

4 Flow rate 1ml/min

5 Runtime 10min

Fig: 2.E Chromatogram of Trial

Result: Because of low theoretical plates it was not selected as system

suitable peak and further trials were conducted.

2.4.5. Optimized chromatographic conditions

A mixture of methanol, water and acetonitrile (50:30:20v/v) with C18

column was proved to be the most suitable of all the combinations since the

chromatographic peak obtained was better defined and resolved and almost

66

free from tailing. Chromatographic conditions as optimized above were

shown in Table 2.5 These optimized conditions were followed for the

determination of anastrozole in bulk samples and its combined tablet

formulations. The chromatograms of blank, standard and sample were shown

in Figure 2.F, 2.G and 2.H.

Table: 2.5 Optimized method conditions for anastrozole

Mobile phase Methanol, water and acetonitrile(50:30:20v/v)

Pump mode Isocratic

Mobile phase PH 6.1

Diluents Mobile phase

Column C18

Column Temp Ambient

Wavelength 218nm

Injection Volume 20 μl

Flow rate 1.0ml/min

Run time 10min

Retention Time 7.045

67

Figure: 2.F: Blank chromatogram

Fig: 2.G: Chromatogram of standard solution

68

Fig: 2.H: Chromatogram of anasrozole

2.5. VALIDATION OF THE PROPOSED METHOD

The proposed method was validated [18-21] 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. System Suitability

System suitability tests were carried out on freshly prepared standard

stock solutions of anastrozole and it was calculated by determining the

standard deviation of anastrozole standards by injecting standards in five

replicates at 6 minutes interval and the values were recorded.

69

2.5.2. Specificity

The selectivity of an analytical method is its ability to measure

accurately and specifically the analyte of interest in the presence of

components that may be expected to be present in the sample matrix. If an

analytical procedure is able to separate and resolve the various components

of a mixture and detect the analyte qualitatively the method is called

selective. It has been observed that there are no peaks of diluents and

placebo at main peaks. Hence, the chromatographic system used for the

estimation of anastrozole is very selective and specific. Specificity studies

indicating that the excipients did not interfere with the analysis. The standard

solution show symmetric peak with retention time of 7.04 min with neat base

line and without any interference of excepients. This indicates that the

proposed method is specific. The specificity results are shown in Table 2.6.

Table 2.6: Specificity study

Name of the solution Retention Time

Blank No peaks

Anastrozole 7.045min

2.5.3. Linearity

Into a series of five standard test tubes, varying amount of standard

stock solution of anastrozole was taken and made up to various

concentrations of 2-10 µg/ml. 20μl was injected from each flask. The peak

area responses of the solutions were recorded at 218nm. The plot of peak

area versus the respective concentrations of anastrozole were found to be

linear in the range of 20-140 µg/ml with coefficient of correlation (r = 0.999)

70

and regression equation of Y= 39419X + 2358 as shown in Fig 2.H. Results

were shown in table 2.7.

Table 2.7: Linearity results

Level

Concentration of

In Anastrozole µg/ml

Peak area

Level -1 2 80548.6

Level -2 4 161967.5

Level -3 6 237749.8

Level -4 8 316754.1

Level -5 10 397347.0

Range: 2µg/ml to

10µg/ml

Slope

Intercept

Correlation coefficient

39419.17

2358.38

0.999

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

Fig: 2.H: calibration curve of linearity

0

100000

200000

300000

400000

500000

0 2 4 6 8 10 12

a

r

e

a

conc. in ppm

Anastrozole

71

2.5.4. 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 and inter day precision.

2.5.4.A. Intra-day precision

To study the intraday precision, six replicate standard solutions (4µg/ml) of

anastrozole were injected. The percent relative standard deviation (% RSD)

was calculated and it was found to be 0.588, which were well within the

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

are shown in Table 2.8.

Table 2.8: Intra-day precision results

Sample

Concentration

(in µg/ml

Injection

number

Area

R.S.D

(Acceptance

criteria ≤2.0%)

Anastrozole

4

1 162742.2

0.588

2 160814.7

3 161458.2

4 160535.0

5 162189.0

6 160395.3

72

2.5.4. B. Inter Day precision

To study the inter day precision, six replicate standard solution

(4µg/ml) anastrozole were injected on third day of sample preparation. The

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

to be 0.918 which were well within the acceptable criteria of not more than

2.0. Results of system precision studies are shown in Table 2.9

2.5.5. Ruggedness

Inter day variations were performed by using six replicate injections of

standard and sample solutions of concentrations which were prepared and

analyzed by different analyst on three different days over a period of one

week. Ruggedness also expressed in terms of percentage relative standard

deviation.

Table 2.9: Inter Day precision

Sample

Concentration

(in µg/ml

Injection

number

Area

R.S.D

(Acceptance

criteria

≤2.0%)

Anastrozole

4µg/ml

1 159234.1

R.S.D = 0.918

2 155217.2

3 158056.7

4 158956.3

5 157297.9

6 157360.7

73

Table: 2.10: Ruggedness results

Sample

Concentration

(in µg/ml

Injection

number

Area

R.S.D

(Acceptance

criteria ≤2.0%)

Anastrozole

4µg/ml

1 162478

R.S.D = 0.17

2 162671

3 162730

4 162728

5 162148

6 162154

2.5.6. Robustness

Robustness was carried out by varying two parameters from the

optimized chromatographic conditions.

Table: 2.11: Robustness results

S.NO Parameter Change Area % Of

Change

1 Standard ……….. 161967 …………

2 Mobile

phase

Methanol:water:Acetonitrile

(55:30:15)

162459 0.3

3 Methanol:water:Acetonitrile

(45:30:25)

161545 0.27

4 Wavelength 220nm 158871 1.9

5 216nm 159862 1.3

6 Mobile

phase PH

6.3 158880 1.9

7 5.9 160279 1.05

74

2.5.7. Accuracy:

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% levels of standard

4µg/ml. 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 2.12. Satisfactory recoveries ranging from 99.5

to 101.2 were obtained by the proposed method. The values of recovery

justify the accuracy of the method. The percentage recovery values were

obtained within the standard limit which confirms that the method is accurate

and free from any positive or negative interference of the excipients. This

indicates that the proposed method was accurate.

Table: 2.12 Recovery results

% of

Recovery

Anastrozole

Target Conc.,

(µg/ml)

Spiked conc.,

(µg/ml)

Final Conc.,

(µg/ml)

Conc., Obtained

% of Assay

50%

4µg/ml 2µg/ml 6µg/ml 5.97 99.5

4µg/ml 2µg/ml 6µg/ml 6.03 100.5

4µg/ml 2µg/ml 6µg/ml 6.01 100.2

100%

4µg/ml 4µg/ml 8µg/ml 7.94 99.25

4µg/ml 4µg/ml 8µg/ml 8.01 100.03

4µg/ml 4µg/ml 8µg/ml 7.96 99.5

4µg/ml 6µg/ml 10µg/ml 10.05 100.5

75

150% 4µg/ml 6µg/ml 10µg/ml 10.12 101.2

4µg/ml 6µg/ml 10µg/ml 10.01 100.1

2.5.8. 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 0.05 µg/ml dilution,

Peak was not clearly observed. So it confirms that 0.05µg/ml is limit of

detection. And limit of quantification found to be 0.165µg/ml.

Table 2.13: LOD and LOQ results

Parameter Measured volume

Limit of Quantification 0.165µg/ml

Limit of Detection 0.05µg/ml

2.5.9. Formulation:

For carrying out assay, 10mg of the anastrozole drug was accurately

weighed and transferred in to a 10ml volumetric flask. Then dissolved in the

solvent was sonicated and made up to make with the diluents and filtered.

One ml of this solution is pippetted out in to the 10ml of volumetric flask and

made up to the mark with the diluents and prepared 10µg/ml solution.

Drug Dosage

form

Brand

name

Sample

concentration

Amount

estimated

% of

Assay

Anastrozole Tablets Redest-

1mg

10µg/ml 0.99% 99.45

76

2.6. DISCUSSION ON THE RESULTS

The results of optimization studies showed that the mixture of

methanol, water and acetonitrile (50:30:20) proportions gave satisfactory

results. The pH of mobile phase is 6.1. The optimized flow rate was found to

be 1.0ml/min. The retention time under these chromatographic conditions

was found to be 7.045minutes with total run time of 10minute. No

endogenous interfering peaks were observed in sample solution confirming

the specificity of the method. Chromatograms of standard solution (9µg/ml)

and blank solution were shown in Figure 2.F, 2.G. System suitability

parameters such as tailing/ asymmetry factor and relative standard deviation

(RSD) were found to be well within acceptable limits (Table 2.5).

A six point calibration curve was constructed and found to be

linear (Fig-2.H). The peak area of the drug was linear in the range of 2-

10µg/ml. The average areas for each of the concentration obtained were

plotted against the concentration of the analyte. The correlation coefficient

for the data was calculated as 0.999 for anastrozole indicating a strong

correlation between the concentration and the area under the curve.

Precision was evaluated by carrying out six independent sample preparation

of a single lot of formulation. Percentage relative standard deviation (%RSD)

was found to be less than 2% for within a day ( 0.588) and day to day

(0.918)variations, which proves that method is precise.

The recovery studies were performed by standard addition

method at 50%, 100% and 150%. Known amounts of standard anastrozole

were added to pre-analyzed samples and were subjected to the proposed

HPLC method. The results showed good recoveries ranging from 99.5 to

101.2%. The mean recovery data obtained for each level as well as for all

77

levels combined were within 2.0% of the label claim for the active substance

with an R.S.D. < 2.0%, which satisfied the acceptance criteria set for the study.

The limit of detection (LOD) is the smallest amount of a sample that can be

differentiated from background noise but not quantified. LOD was

determined as the amount for which the signal-to-noise ratio (S/N) was 3:1 by

comparing results from samples of known concentration with results from

blank samples. The limit of quantification (LOQ) was defined as the lowest

concentration of analyte that could be determined with acceptable precision

and accuracy and was established by determining the concentrations of four

spiked calibration standards. The LOD and LOQ were found to be 0.05µg/ml

and 0.165µg/ml respectively.

To evaluate the robustness of the developed RP-HPLC method,

small deliberate variations in the optimized method parameters were done.

The effect of change in flow rate, pH and mobile phase ratio on the retention

time and tailing factor were studied. These parameters were found to

proportionally change which are given in Table 5.8. The sample solution

stored at ambient temperature (20±100C) for two days to evaluate the

stability of the proposed method. The main peak area was studied for the

peak purity (anastrozole peak), thus indicating that the method effectively

separated the degradation products from the anastrozole active ingredient. It

was noticed that solutions were stable for 48 hrs, as during this time the

results did not decrease below 98%. This denotes that anastrozole was stable

and the standard and sample solutions for at least two days at ambient

temperature. The validated method was applied for the assay of commercial

tablets (Redest-1mg) containing anastrozole. The results presented good

agreement with the labeled content.

78

The method was found to be accurate, reproducible, and specific and

rapid. No interfering peaks were observed at the elution times of anastrozole.

The method may be applied for the estimation of anastrozole in bulk and in

pharmaceutical formulations without interference and with good sensitivity.

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