CHAPTER IV

SPECTROPHOTOMETRIC AND ULTRA PERFORMANCE LIQUID CHROMATOGRAPHIC ASSAY OF CLOMIFENE CITRATE

106

Section 4.0

DRUG PROFILE AND LITERATURE SURVEY

4.0.1 DRUG PROFILE

Clomifene citrate (CMC) is chemically known as 2-[p-(2chloro-1,2-

diphenylvinyl)phenoxy] triethylamine citrate (1:1) [1]. CMC is a white crystalline

solid. It has the molecular formula of C26H28ClNO • C6H8O7 and its molecular

mass is 598.10 g mol-1. The chemical structure of CMC is as shown below:

CMC is slightly soluble in water and alcohol, practically insoluble in

diethyl ether, and is freely soluble in glacial acetic acid, methanol, acetonitrile,

dichloromethane. It was first synthesized in 1956 by Frank Palopoli et al., of

Merrell chemistry department and approved for clinical use in 1967.

Clomifene is a non-steroidal compound which has both estrogenic and

antiestrogenic effect. It is a mixture of the Z isomer and E isomer and contains not

less than 30 % and not more than 50% of the Z isomer [2]. CMC is primarily used

for the treatment of anovulatory infertility [3]. It has also been used in the

treatment of male infertility [4], pubertal gynecomastia and seizure disorders [5].

Other uses of Clomifene are: as an adjunct in in-vitro fertilization, embryo transfer

and intrauterine insemination [6]. Clomifene exerts its therapeutic effects by

increasing the output of pituitary gonadotrophic hormones by blocking the binding

of endogenous estrogen to hypothalamic and pituitary estrogen receptors [7].

107

4.0.2 LITERATURE SURVEY OF ANALYTICAL METHODS FOR

CLOMIFENE CITRATE

The drug is official in the United States Pharmacopeia [2] and British

Pharmacopeia [8], which describes high performance liquid chromatographic

methods for its assay.

4.0.2.1 Spectrophotometric methods

Not many methods are found in the literature for the determination of

CMC in pharmaceuticals. Hewala [9] has reported three methods one by

derivative UV-spectrophotometry and the two by visible spectrophotometry. In

the first visible spectrophotometric method, the colored ion-pair formed with

methyl orange was extracted in to chloroform and absorbance measured at 420

nm; the second is based on the reaction of CMC with citric acid and acetic

anhydride in CHCl3 forming a blue colored chromogen measurable at 605 nm.

CMC is found to react with I2, ammonium molybdate or phosphomolybdic acid to

form molecular complex, the latter serving as a basis for the determination of drug

by three indirect methods [10]. A method based on the formation of a colored

radical anion (λmax: 460 nm) when CMC is treated with 2,3-dichloro-5,6-dicyano-

p-benzoquinone (DDQ) in chloroform medium has been reported by Mallikarjuna

Rao et al [11]. Ammonium reineckate is reported to form an insoluble ion-pair

complex with CMC [12]. The precipitate after washing with water and filtering

was dissolved in acetonitrile and absorbance measured at 509 nm offering a

method for the determination of CMC in the 0.2-1.8 mg ml-1 range.

4.0.2.2 Chromatographic methods

A close examination of the chromatographic methods for CMC reveals

that the reported methods deal with either separation of the E and Z isomers,

determination in body fluids or some interactions. A normal phase HPLC method

was used to separate cis and trans- isomers by Harman et al [13]. The

determination of CMC in biological matrices by liquid-phase extraction combined

with photochemical fluorescence HPLC has recently been reported by Xiaohong

et al [14]. A few more HPLC methods have been reported for the analysis of the

two clomifene isomers in biological matrices [15-18]. A sensitive and selective

LC-MS method [19] has been reported for the simultaneous determination of Z

and E isomers in plasma of patients undergoing treatment for the induction of

ovulation. In addition, high performance affinity chromatography [20] has been

108

used to examine the binding interaction of the isomers of CMC with human serum

albumin (HSA). In a similar study by the same authors [21], interactions of cis and

trans-clomifene with HAS in the presence of β-cyclodextrin has also been

reported.

4.0.2.3 Other techniques

Several other techniques including nmr spectroscopy [22], resonance Rayleigh

scattering spectrometry [23, 24], conductometry [12], potentiometry [25] and

capillary electrophoresis [26] are found in the literature for the assay of drug in

pharmaceuticals.

The only UV-spectrophotometric method [9] reported is applicable to

combined dosage form. The method based on ion-pair formation [9] requires strict

pH control and extraction step. Six procedures [10, 12] are cumbersome and

involve precipitation and filtration steps and are prone to loss of analyte there by

affecting the accuracy of the methods. The method based on charge-transfer

complexation reaction and involving the use of DDQ is handicapped by longer

contact time, narrow linear dynamic range and poor sensitivity. It is also apparent

from the literature survey that no chromatographic method has ever been applied

to determine CMC in pharmaceutical dosage forms.

Keeping the above points in view, the author has developed two UV and

five visible spectrophotometric methods, the latter being based on ion-pair

reaction without extraction, and charge-transfer complexation reactions. Also,

ultra performance liquid chromatography (UPLC) has been applied for the first

time to the determination of CMC in pharmaceuticals. The details about the

method development and validation of these methods are presented in this chapter.

109

Section 4.1

SIMPLE UV-SPECTROPHOTOMETRIC METHODS FOR THE

DETERMINATION OF CLOMIPHENE CITRATE IN

PHARMACEUTICALS

4.1.1 INTRODUCTION

A smart profile and utilization of UV-spectrometry in different assay have

been presented in Section 3.2. From the literature survey presented Section 4.0 it

is evident that one derivative UV-spectrophotometric method [9] has been

reported for the quantification of CMC.

In the literature, no stability-indicating UV-spectrophotometric methods

have ever been reported for the assay of CMC. In the present Section (4.1), two

simple, inexpensive, accurate, reproducible, and stability-indicating UV-

spectrophotometric methods for CMC are described. The methods are based on

the measurement of absorbance of CMC solution either in 0.1 M H2SO4 at 290 nm

in method A, or 0.1 M HCl at 289 nm in method B. Besides, the methods were

used to study the degradation of the drug under stress conditions as per the ICH

guidelines [27].

4.1.2 EXPERIMENTAL

4.1.2.1 Apparatus

The instrument used for absorbance measurement is the same as

described in Section 3.2.2.1.

4.1.2.2 Materials

All chemicals used were of analytical reagent grade. Doubly-distilled

water was used to prepare solutions wherever required. Hydrogen peroxide

(H2O2), sulphuric acid (H2SO4), hydrochloric acid (HCl) and sodium hydroxide

(NaOH) were purchased from Merck (Mumbai, India). Pure CMC

(Pharmaceutical grade) sample was kindly provided by Jubiliant Life Sciences,

Ltd. Nanjangud, Mysore, India, as a gift and used as received. Two brands of

tablets, namely, Siphene-25 and Siphene-100 (both from Maneesh

Pharmaceuticals Ltd., Mumbai) were obtained from the commercial sources.

Sulphuric acid (H2SO4, 0.1 M): Prepared by diluting concentrated acid (Merck,

Mumbai, India. Sp.gr.1.84) with water.

Hydrochloric acid, hydrogen peroxide, sodium hydroxide solutions

required for degradation study were prepared as described under Section 3.2.2.

110

Standard drug solution

Standard drug solutions of 100 µg ml-1 CMC were prepared by dissolving

accurately weighed 10 mg of pure drug in 0.1 M H2SO4 in method A, and 0.1 M

HCl in method B, separately.

4.1.2.3 General procedures

Method A

Varying aliquots (0.2, 0.5, 1.0, 2,0, 3.0, 4.0, 5.0 and 6.0 ml) of working

standard solution corresponding to 2-60 µg ml-1 CMC were taken in a series of 10

ml volumetric flasks and volume was made upto mark with 0.1 M H2SO4. The

absorbance of each solution was measured at 290 nm against 0.1 M H2SO4.

Method B

Into a series of 10 ml calibration flasks, aliquots of CMC standard solution

(100 µg ml-1) equivalent to 2-60 µg ml-1 CMC were accurately transferred and

volume was made upto mark with 0.1 M HCl. The absorbance of each solution

was measured at 289 nm versus 0.1 M HCl.

In both the cases, calibration curves were prepared and the concentration of

the unknown was read from the respective calibration curve or computed from the

regression equation derived using the Beer’s law data.

4.1.2.4 Procedure for tablets

Weighed amount of tablet powder equivalent to 10 mg of CMC was

transferred into a 100 ml volumetric flask. The content was shaken well with

about 50 ml of 0.1 M H2SO4/ 0.1 M HCl for 20 min. The mixture was diluted to

the mark with the respective acid. It was filtered using Whatman No 42 filter

paper. First 10 ml portion of the filtrate was discarded and a subsequent portion

was subjected to analysis by following the procedure described earlier.

Placebo blank analysis

A placebo blank of the composition: talc (75 mg), starch (85 mg), acacia

(80 mg), methyl cellulose (90 mg), sodium citrate (80 mg), magnesium stearate

(90 mg) and sodium alginate (75 mg) was made. By taking 20 mg, its solution

was prepared as described under “procedure for tablets” and then subjected to

analysis.

Procedure for synthetic mixture analysis

To 20 mg of the placebo blank of the composition described above, 10 mg

of CMC was added and homogenized, transferred to 100 ml calibrated flask and

111

the solution was prepared as described under “procedure for tablets” Then the

resulting solution were subjected to analysis using the procedures described

above. The analysis was done to study the interferences of excipients such as talc,

starch, acacia, methyl cellulose, sodium citrate, magnesium stearate and sodium

alginate in the assy.

Forced degradation study (Stability study)

In both the methods, a 3 ml aliquot of 100 µg ml-1 CMC was taken (in

triplicate) in a 10 ml volumetric flask and mixed with 5 ml of 5 M HCl (acid

hydrolysis) or 5 M NaOH (alkaline hydrolysis) or 5% H2O2 (oxidative

degradation) and boiled for 2 h at 80 °C in a hot water bath. The solution was

cooled to room temperature and diluted to the mark with 0.1 M HCl after

neutralization with base/acid. In thermal degradation, solid drug was kept in Petri

dish in oven at 100 °C for 24 h. After cooling to room temperature, 100 µg ml-1

CMC solutions in 0.1 M HCl/H2SO4 were prepared separately and absorbance

measured. For UV degradation study, the stock solutions of the drug (100 µg ml-1)

were exposed to UV radiation of wavelength 254 nm and of 1.2K flux intensity

for 48 h in a UV chamber. The solutions after dilution with either 0.1 M HCl or

0.1 M H2SO4 were assayed as described above.

4.1.3 RESULTS AND DISCUSSION

4.1.3.1 Spectral characteristics

The absorption spectra of 10 µg ml-1 CMC solution in 0.1 M H2SO4

(method A) and 30 µg ml-1 CMC solution in 0.1 M HCl (method B) were recorded

between 200 and 400 nm and showed absorption maxima at 290 and 289 nm, for

method A and method B, respectively. At these wavelengths, 0.1 M H2SO4 and

0.1 M HCl had insignificant absorbance. Therefore, the analysis of CMC was

carried out at 290 and 289 nm, for method A and method B, respectively (Figure

4.1.1).

4.1.3.2 Forced degradation study

The absorption spectra of the CMC solutions in 0.1 M H2SO4 and 0.1 M

HCl treated with acid, base hydrolysis, hydrogen peroxide, dry heat and UV

radiation were run in the range of (200-400 nm). The degradation was evaluated

based on the comparison of the UV spectra of “stressed CMC samples” with that

of the “standard CMC solution” [28].

112

The UV-spectra of 30 µg ml-1 CMC each in 0.1 M H2SO4 and 0.1 M HCl

after forced degradation are shown in Figure 4.1.2 to Figure 4.1.6. The drug was

found to undergo slight degradation in method B and remained intact in method A

after acid hydrolysis (Figure 4.1.2). Base hydrolysis resulted in significant

degradation in both methods (Figure 4.1.3). The absorption spectra of CMC

solution subjected to H2O2 showed that the drug experienced slight degradation in

method A and significant degradation in method B (Figure 4.1.4) The drug did

not undergo degradation after exposure to heat and light as revealed by the

absorption spectra which are similar to that of unstressed CMC solution (Figure

4.1.5 and Figure 4.1.6).The results of this are presented in Table 4.1.0.

(a) (b)

Figure 4.1.1 Absorption spectra of 10 µg ml-1 CMC; (a) in 0.1 M H2SO4; (b) in 0.1 M HCl.

4.1.3.3 Method validation

Linearity and sensitivity

The regression parameters calculated from the calibration graphs data, are

presented in Table 4.1.2. Beer’s law was obeyed over the concentration ranges

shown in Table 4.1.2, and the linearity of calibration graphs (Figure 4.1.7) was

demonstrated by the high values of the correlation coefficient (r) and the small

values of the y-intercepts of the regression equations The molar absorptivity,

Sandell sensitivity values of both methods are also shown in Table 4.1.2.The

limits of detection and quantification were calculated as per the current ICH

guidelines [27] and are presented in Table 4.1.2.

113

Table 4.1.1 Results of degradation study

Degradation condition % Degradation Method A Method B

No degradation ( control) Zero Zero

Acid hydrolysis (5 M HCl , 80°C, 2 hours)

Zero 4.4

Base hydrolysis (5 M NaOH , 80°C, 2 hours)

Total Total

Oxidation (5% H2O2 , 80°C, 2 hours) 11.1 Total

Water hydrolysis (water , 80°C, 2 hours)

Thermal (105°C, 4 hours) Zero Zero

Photolytic (1.2 K flux, 48 hours) Zero Zero

(a) (b)

Figure 4.1.2 Absorption spectra of 30 µg ml-1CMC after acid hydrolysis (5M HCl)(a) in 0.1 M H2SO4;(b)in 0.1 M HCl .

(a)

114

(b)

Figure 4.1.3 Absorption spectra of 30 µg ml-1CMC after base hydrolysis (5M NaOH)(a) in 0.1 M H2SO4;(b)in 0.1 M HCl .

(a)

(b)

Figure 4.1.4 Absorption spectra of 30 µg ml-1CMC after oxidative degradation

(5% H2O2) a) in 0.1 M H2SO4;(b)in 0.1 M HCl .

115

(a)

(b)

Figure 4.1.5 Absorption spectra of 30 µg ml-1CMC after thermal degradation

(105 oC for 4 hours)(a) in 0.1 M H2SO4;(b)in 0.1 M HCl.

Precision and accuracy

Accuracy was evaluated as percentage relative error between the

measured and taken concentrations of CMC (RE %). The results, compiled in

Table 4.1.3, show that the accuracy is good for both methods. Precision of the

methods was calculated in terms of intermediate precision (intra-day and inter-

day). Three different concentration of CMC (within the working limits) were

analyzed in seven replicates during the same day (intra-day precision) and five

consecutive days (inter-day precision). RSD (%) values (Table 4.1.3) of the intra-

day and inter-day studies showed that the precision was good for the both

methods.

116

(a)

(b)

Figure 4.1.6 Absorption spectra of 30 µg ml-1CMC after photo degradation (1200

flux hours)(a) in 0.1 M H2SO4;(b)in 0.1 M HCl .

Selectivity

A systematic study was performed to determine the effect of matrix by

analyzing the placebo blank and synthetic mixture containing CMC. A placebo

blank was prepared by following the procedure described in Section 4.1.2.3 and

then subjected to analysis. The absorbance of the placebo solution in each case

was almost equal to the absorbance of the blank which revealed no interference.

To assess the role of the inactive ingredients on the assay of CMC, a synthetic

mixture was separately prepared by adding 10 mg of CMC to the placebo. The

117

drug was extracted and solution prepared as described under the general procedure

for tablets. The solutions were analyzed by following the recommended

procedures. The percentage recovery values of CMC obtained from this study

were in the range from 99.32 to 102.54. This unequivocally demonstrated the non-

interference of the inactive ingredients in the assay of CMC. Further, the slopes of

the calibration plots prepared from the synthetic mixture solutions were about the

same as those prepared from pure drug solutions.

Method A Method B

Figure 4.1.7. Calibration curves.

Table 4.1.2. Sensitivity and regression parameters

Parameter Method A (0.1 M H2SO4)

Method B (0.1 M HCl)

max, nm 290 289 Linear range, µg ml-1 2.0 – 60.0 2.0 – 60.0

Molar absorptivity(ε), l mol-1 cm-1 1.0 × 104 1.0 × 104

Sandell sensitivity, µg cm-2 0.0592 0.0568 Limit of detection (LOD), g ml-1 0.80 0.94 Limit of quantification (LOQ), g ml-1 2.42 2.84 Regression equation, Y* Intercept (a) 0.0104 0.0012 Slope (b) 0.0178 0.0178 Standard deviation of a (Sa) 0.1262 0.0441 Standard deviation of b (Sb) 0.0070 0.0025 Regression coefficient (r) 0.9998 0.9999

*Y=a+bX, Where Y is the absorbance; X is concentration in µg ml-1; a intercept and b slope.

0

0.2

0.4

0.6

0.8

1

1.2

0 10 20 30 40 50 60

Abso

rban

ce

concentration of CMC, µg ml-1

0

0.2

0.4

0.6

0.8

1

1.2

0 10 20 30 40 50 60

Abso

rban

ce

Concentration of CMC, µml-1

118

Table 4.1.3. Results of intra-day and inter-day accuracy and precision study

%RE. Percent relative error, %RSD. Relative standard deviation

Ruggedness

Method ruggedness was demonstrated having the analysis done by four

analysts, and also by a single analyst performing analysis with four different

cuvettes in the same laboratory. Intermediate RSD (%) in both instances were in

the range 1.28-2.85% indicating acceptable ruggedness. The results are presented

in Table 4.1.4.

Table 4.1.4 Results of method ruggedness study expressed as intermediate

precision, RSD (%)

Method CMC taken, µg ml-1

Inter-analysts RSD (%) (n=4)

Inter-cuvettes RSD (%) (n=4)

A 20.0 30.0 40.0

1.28 2.06 1.62

2.85 2.46 2.06

B 20.0 30.0 40.0

1.75 1.98 2.05

2.25 2.33 2.10

Analysis of tablets

The proposed methods were applied for the quantification of CMC in

commercial tablets. The results were compared with those of official method [2]

in which the sample was chromatographed on a column (4.6 mm × 25 cm)

Method CMC taken, µg ml-1

Intra-day accuracy and precision

(n=7)

Inter-day accuracy and precision

(n=5) CMC found, µg ml-1

%RE %RSD CMC found

µg ml-1 %RE %RSD

A

20.0 30.0 40.0

20.37 30.30 39.48

1.76 1.01 1.29

0.97 1.53 1.16

20.46 30.35 39.62

1.84 1.66 1.32

1.84 1.55 1.87

B 20.0 30.0 40.0

20.31 30.49 40.27

0.88 1.10 1.73

1.54 1.63 0.70

20.42 30.53 40.22

1.12 1.33 1.92

1.18 1.60 1.46

119

containing packing L26 with a mobile phase consisting of methanol, water and

triethylamine (55:45:0.3), at a flow rate of 1.0 ml min-1 and the UV-detection

being set at 233 nm. The assay was performed for two different brands of tablets

containing 25 and 100 mg of active ingredient (Siphene-25 and Siphene-100) as

described earlier. Statistical analysis of the results did not detect any significant

difference between the performance of the proposed methods and reference

method with respect to accuracy and precision as revealed by the Student’s t-value

and variance ratio F-value [29]. The results of this study are presented in Table

4.1.5.

Table 4.1.5 Results of analysis of tablets by the proposed methods and statistical

comparison of the results with the official method

*Mean value of five determinations.

Recovery study

To further assess the accuracy of the methods, recovery experiments were

performed by applying the standard-addition technique. The recovery was

assessed by determining the agreement between the measured standard

concentration and added known concentration to the sample. The test was done by

spiking the pre-analyzed tablet powder with pure CMC at three different levels

(50, 100 and 150 % of the content present in the tablet powder (taken) and the

total was found by the proposed methods. Each test was repeated three times. In

all the cases, the recovery percentage values ranged between 99.06 and 100.76%

with relative standard deviation in the range 0.58-1.72%. Closeness of the results

to 100% showed the fairly good accuracy of the methods. The results are shown in

Table 4.1.6.

Tablet brand Name#

Nominal amount (mg/tablet)

Found* (Percent of label claim ± SD) Official method Method A Method B

Siphene

25

99.36±0.68

100.2±1.17 t=2.25 F=2.96

99.65±1.46 t=1.03 F=4.60

Siphene 100 100.92±0.72 100.1±0.89 t=2.52 F=1.52

99.96±0.89 t=2.34 F=2.82

120

Table 4.1.6. Results of recovery study via standard-addition method.

*Mean value of three determinations

Tablets studied

Method A Method B

CMC in tablet, µg ml-1

Pure CMC added, µg ml-1

Total found, µg ml-1

Pure CMC recovered (Percent±SD*)

CMC in tablet, µg ml-1

Pure CMC added, µg ml-1

Total found, µg ml-1

Pure CMC recovered (Percent±SD*)

Siphene-25

20.04 20.04 20.04

10.0 20.0 30.0

29.95 40.10 49.66

99.17±1.72 100.33±0.98 98.76±1.06

19.93 19.93 19.93

10.0 20.0 30.0

29.96 39.77 49.64

100.37±0.83 99.23±1.30 99.06±0.78

Siphene-100

20.02 20.02 20.02

10.0 20.0 30.0

30.07 40.17 49.86

100.50±0.72 100.76±1.38 99.46±1.06

19.96 19.96 19.96

10.0 20.0 30.0

29.86 39.94 50.08

99.09±0.58 99.92±1.30 100.43±0.65

121

Section 4.2

APPLICATION OF EXTRACTION-FREE ION-PAIR COMPLEXATION

REACTION FOR THE SPECTROPHOTOMETRIC DETERMINATION

OF CLOMIPHENE CITRATE IN PHARMACEUTICALS

4.2.1 INTRODUCTION

According to IUPAC [30] ion-pair is a pair of oppositely charged ions held

together by coulombic attraction without formation of a covalent bond. Modern

pharmaceutical analysis demands analytical methods for the determination of

desired component when it is present in a complex dosage formulation followed

by its instrumental determination. Extractive spectrophotometry is placed at the

top for such type of analysis because it can be applied to the determination of an

individual component in the presence of routine excipients and filling materials.

This aspect of spectrophotometric analysis is of great interest since it offers

distinct possibilities for the assay of a particular component in a complex dosage

formulation [31].

In an extractive spectrophotometric analysis, ion-association is a chemical

reaction whereby ions of opposite electrical charge come together in solution to

form a distinct chemical entity. Ion-pairs are formed when a cation and anion,

present in aqueous phase (aq), combine together [32-35] and these complexes are

coloured when extracted into a organic solvent (org).

An+(aq) + Bm-

(aq) AB(n-m)+(org)

A hypothesis based on the possible role of solvated ion pair species in enhancing

the extractive process, proposed by Hull [36] and Higuchi [37], assumes that the

free energy involved in the transference of the ionic components from the water

phase to form simple ion pairs in the organic phase is predominant. Many ion-pair

extractive spectrophotometric methods have earlier been used in the assay of

many pharmaceutically important substances and include tilidine [38], doxazosine

mesylate [39], cyproheptadine hydrochloride [40], enoxacine [41], enrofloxacin

and pefloxacin [42], promethazine theoclate [43], maprotilin hydrochloride [44],

berberine and benzethonium [45], lamotrigine [46, 47], quetiapine fumarate [48]

and alfuzosin hydrochloride [49], to mention a few. In these cases, an ion-pair is

formed between a protonated basic compound and an anionic dye. At a specific

pH, the ion-pair, which is immiscible with water, is extracted into an organic

solvent and the concentration of the drug is determined spectrophotometrically.

122

Though, ion-pair extractive spectrophotometry has several advantages, it has some

difficulties and inaccuracies arising from incomplete extraction or the formation

of emulsions between the organic solvent and the basic compound containing

solution. In response to this problem, extraction-free ion-pair spectrophotometry is

gaining importance in the field of pharmaceutical analysis. Few articles were

published for the analysis of pharmaceutical compounds through ion-pair

formation without involving extraction [50-55].

In the literature survey presented in Section 4.0.2, an extractive

spectrophotometric method for the determination of CMC through the ion pair

complex formation with methyl orange as a reagent [9] has been reported. The

method requires strict pH control, tedious and time-consuming extraction step and

is prone to inaccuracy due to incomplete extraction of the analyte. In this Section

(4.2), three simple extraction-free spectrophotometric methods using three

sulphonthalein dyes are described. The methods are based on formation of yellow

ion-pairs between clomiphene and three sulphonthalein dyes; bromothymol blue

(BTB) (method A), bromocresol green (BCG) (method B), and bromocresol

purple (BCP) (method C), in dichloromethane medium followed by absorbance

measurement at 420, 415 and 425 nm, respectively. The method development,

validation and its applications are presented in this Section (4.2).

4.2.2 EXPERIMENTAL

4.2.2.1 Instrument

The instrument is the same that was described in Section 2.2.2.1.

4.2.2.2 Reagents and materials

All reagents were of analytical reagent grade and HPLC grade organic solvents

were used throughout the investigation.

Bromothymol Blue (0.1%), Bromocresol green (0.05%) and Bromocresol

purple (0.1%): The solutions of bromothymol blue (BTB, Loba Chemie, Mumbai,

India), bromocresol green (BCG, Merck, Mumbai, India) bromocresol purple

(BCP, Loba Chemie, Mumbai, India) were prepared in dichloromethane (Merck,

Mumbai, India, Sp. gr. 1.32).

Standard CMC solution: A stock standard CMC solution (100 µg ml-1) was

prepared by dissolving 10 mg of pure CMC in dichloromethane and diluting to the

mark in a 100 ml calibrated flask with dichloromethane. The working standard

solutions of 50 µg ml-1 (for method A) and 40 µg ml-1 (for method B and method

123

C) were then prepared by suitable dilution of the stock solution with

dichloromethane.

The pharmaceutical preparations used in this study were the same mentioned in

previous section.

4.2.2.3 Assay procedures

Method A (using bromothymol blue)

Different aliquots (0.2-3.5 ml) of a standard CMC solution (50 µg ml-1)

were transferred into a series of 5 ml calibrated flasks using a micro burette and to

each flask was added 1 ml of 0.1% BTB solution. The mixture was diluted to the

volume with dichloromethane and mixed well. The absorbance of each solution

was measured at 420 nm against a reagent blank after 5 min.

Method B (using bromocresol green)

Different aliquots (0.2-3.5 ml) of a standard CMC (40 µg ml-1) solution

were transferred into a series of 5 ml calibrated flasks, as described above. To

each flask was added 1 ml of 0.05% BCG solution and diluted to the volume with

dichloromethane and mixed well. The absorbance of each solution was measured

at 415 nm against a reagent blank after 5 min.

Method C (using bromocresol purple)

Different aliquots (0.2-3.5 ml) of a standard CMC (40 µg ml-1) solution

were transferred into a series of 5 ml calibrated flasks, as described above. To

each flask was added 1 ml of 0.1% BCP solution and diluted to the volume with

dichloromethane and mixed well. The absorbance of each solution was measured

at 425 nm against a reagent blank after 5 min.

Procedure for tablets

Ten tablets were weighed accurately and ground into fine powder. An

amount of the powder equivalent to 10 mg of CMC was weighed into a 100 ml

calibrated flask containing about 60 ml of dichloromethane. The solution was

shaken thoroughly for about 15-20 min, diluted to the mark with dichloromethane,

and filtered using a Whatman No. 42 filter paper. First 10 ml portion of filtrate

was discarded and subsequent portions were subjected to analysis by the

procedure described above after dilution to 50 and 40 µg ml-1 CMC with

dichloromethane.

124

Placebo blank analysis

A placebo blank of the composition: talc (75 mg), starch (85 mg), acacia

(80 mg), methyl cellulose (90 mg), sodium citrate (80 mg), magnesium stearate

(90 mg) and sodium alginate (75 mg) was made. By taking 20 mg of it, solution

was prepared as described under “Procedure for tablets”. The analyses were

performed using the procedures described above.

Procedure for synthetic mixture analysis

To 20 mg of placebo blank of the composition described above, 10 mg of

CMC was added and homogenized, transferred to 100 ml calibrated flask and the

solution was prepared as described under “Procedure for tablets”. Then the

resulting solution after appropriate dilution was subjected to analysis using the

procedure described above.

4.2.3 RESULTS AND DISCUSSION

4.2.3.1 Absorption spectra

The absorption spectra of the ion-pair complexes, formed between CMC

and each of BTB, BCG and BCP recorded at 360-500 nm against the respective

blank solution are shown in Figure 4.2.1. The yellow ion-pair complexes showed

maximum absorbance at 420, 415 and 425 nm for CMC-BTB, CMC-BCG and

CMC-BCP, respectively. The measurements were thus made at these wavelengths.

4.2.3.2 Reaction pathway

Chemically, the structure of CMC features its basic nature. This structure

suggests the possibility of utilizing an anionic dye as chromogenic reagent. In

dichloromethane, CMC does not absorb in the visible region. The dyes employed

have insignificant absorbance at the wavelength of measurement. In contrast,

when a solution of BTB/BCG/BCP in dichloromethane, was added to the drug

solution, an intense yellow coloured product measurable at 420, 415 or 425 nm, in

method A, method B and in method C respectively, was produced immediately

(Fig. 4.2.1). This is due to an opening of lactoid ring and subsequent formation of

quinoid group [56]. It is supposed that the two tautomers are present in

equilibrium but due to strong acidic nature of the sulphonic acid group, the

quinoid body must predominate.

125

Figure 4.2.1 Absorption spectra of: a. CMC-BTB ion-pair complex (20.0 µg ml-1 CMC), b. blank (method A), c. CMC-BCG ion-pair complex (16.0 µg ml-1 CMC), d. blank (method B), e. CMC-BCP ion-pair complex (16.0 µg ml-1 CMC) and f. blank (method C).

Finally, protonated CMC forms ion-pair with the dye. The possible

reaction pathways are shown in Scheme 4.2.1, Scheme 4.2.2 and Scheme 4.2.3.

Anionic dye such as BTB/BCG/BCP forms ion pair complex with positively

charged drug. Each drug-dye ion-pair complex molecule, with two oppositely

charged ions, behaves as a single unit held together by an electrostatic force of

attraction.

4.2.3.3 Method development

Effect of solvent

In order to select the suitable solvent for the formation of ion-pair

complex, the reaction of CMC with BTB, BCG or BCP was studied in different

solvents. Better results were obtained when CMC was dissolved dichloromethane

in all the three methods than other solvents like chloroform, 1,2-dichloroethane,

0

0.1

0.2

0.3

0.4

0.5

0.6

360 380 400 420 440 460 480 500

Abso

rban

ce

Wavelength, nm

a

b0

0.1

0.2

0.3

0.4

0.5

0.6

360 380 400 420 440 460 480 500

Abso

rban

ce

Wavelength, nm

c

d

0

0.1

0.2

0.3

0.4

0.5

0.6

360 380 400 420 440 460 480 500

Abso

rban

ce

Wavelength, nm

e

f

126

acetonitrile or carbon tetrachloride. In the case of dyes, dichloromethane was

preferred to chloroform, acetone, acetonitrile, 2-propanol, 1,2-dichloroethane, 1,4-

dioxane, methanol and ethanol because, as the complex formed in these solvents

had very low sample absorbance values or higher blank absorbance values.

Therefore, dichloromethane was chosen as solvent. C3H7

HO

BrCH3

SO2

O

C3H7OH

Br

C3H7HO

BrCH3

SO3H

C3H7O

Br

C3H7HO

BrCH3

SO3-

C3H7O

Br+ H+

BTB(lactoid ring) (quinoid ring)

C3H7HO

BrH3C SO3

-

C3H7O

Br+ H+

Cl O N

HOO

HO

O OH

OHO

+

CMC

Cl O N

HOO

HO

O OH

OHO

H+ C3H7

HO

BrH3C SO3

C3H7O

Br-

1:1 CMC-BTB complex Scheme 4.2.1. The possible reaction pathway for the formation of CMC-BTB ion-

pair complex.

OHBrBr

HOBr Br

SO2O

(lactoid ring)BCG

OBrBr

HOBr Br

SO3H

(quinoid ring)

OBrBr

HOBr Br

SO3-

+ H+

Cl O N

HOO

OH

O OH

OHO

+

CMC

OBrBr

HOBr Br

SO3-

+ H+Cl O N

HOO

HO

O OH

OHO

H+

OBrBr

HOBr Br

SO3-

1:1 CMC-BCG complexScheme 4.2.2. The possible reaction pathway for the formation of CMC-BCG ion-

pair complex.

127

Cl O N

HOO

HO

O OH

OHO

Cl O N

HOO

HO

O OH

OHO

H+

BrHO

H3C

SO2

O

CH3OH

Br

BrHO

H3C

SO3H

CH3O

Br

BrHO

H3C

SO3-

CH3O

Br+ H+

+

BrHO

H3C

SO3-

CH3O

Br+ H+

BrHO

H3C

SO3

CH3O

Br-

BCP

(lactoid ring) (quinoid ring)

CMC1:1 CMC-BCP complex

Scheme 4.2.3. The possible reaction pathway for the formation of CMC-BCP ion-

pair complex.

Effect of volume of dye and reaction time, and stability of the ion-pair

complex

In order to find out the optimum amount of dye required to obtain

maximum absorbance, experiments were performed separately by measuring the

absorbance of the final solution resulting from the reaction mixture containing a

fixed concentration of CMC and various amounts of the dye. It was found that 1

ml of dye solution (0.1% BTB in method A, 0.05% BCG in method B and 0.1%

BCP in method C) was sufficient to produce maximum and reproducible

absorbance (Fig. 4.2.2). The reaction time or standing time after the addition of

dye was also examined. A 5 min standing time was sufficient for the complete

formation of ion-pair complex. The absorbance of the resulting ion-pair complex

was found to be stable for at least 2 h in method A, 3h in method B and 2.5 h in

method C at room temperature (28±2 °C).

Investigation of composition of ion-pair complex

The composition of the ion-pair complex formed between CMC and

BTB/BCG/BCP was established by applying Job’s method of continuous

variations [57] using equimolar concentrations of CMC and the dye. In method A,

CMC and dye concentration used were 6.79 ×10-5M, 4.20×10-5 M each in method

B, where as 5.61 × 10-5 M each in method C. The experiments were performed by

mixing equimolar solutions of the drug and dye by maintaining the total volume at

5.0 ml. In all the cases, the plot reached a maximum value at a mole fraction of

0.5 which indicated the formation of 1:1 (CMC:dye) complex (Figure 4.2.3), and

128

the results revealed that the formation of ion -pair complex between drug and

reagent followed a 1:1 reaction stoichiometry.

The log Kf values were found to 5.432, 7.562 and 6.225 for method A,

method B, and method C, respectively.

(a) (b)

(c)

Figure 4.2.2 Effect of dye concentration on the formation of ion-pair complex

(a) Method A (20 µg ml-1 CMC, (b) Method B (16.0 µg ml-1 CMC),

and (c) Method C (16.0 µg ml-1 CMC)

4.2.3.4 Method validation

Linearity and sensitivity

Under optimum experimental conditions for CMC determination, the

standard calibration curves for CMC with BTB, BCG and BCP were constructed

by plotting absorbance versus concentration (Figure 4.2.4). The regression

parameters calculated from the calibration graphs data, are presented in Table

4.2.1. Beer’s law was obeyed over the concentration ranges given in Table 4.2.1

and the linearity of calibration graphs was demonstrated by the high values of the

correlation coefficient (r) and the small values of the y-intercepts of the regression

00.10.20.30.40.50.6

0 0.5 1 1.5 2 2.5 3Volume of 0.1% BTB, ml

Abso

rban

ce

0

0.1

0.2

0.3

0.4

0.5

0.6

0 0.5 1 1.5 2 2.5 3

Abso

rban

ce

Volume of 0.05% BCG, ml

00.10.20.30.40.50.6

0 0.5 1 1.5 2 2.5 3

Abso

rban

ce

Volume of 0.1% BCP, ml

129

equations. The apparent molar absorptivities of the resulting colored ion-pair

complexes, Sandell sensitivities, detection and quantification limits were

calculated and shown in Table 4.2.1.

(a) (b)

(c)

Figure 4.2.3 Job’s plots obtained for ion-pair complexes from equimolar

solutions of; (a) CMC & BTB (b) CMC & BCG and (c) CMC &

BCP

Method A Method B

0

0.1

0.2

0.3

0.4

0.5

0.6

0 0.2 0.4 0.6 0.8 1

Abso

rban

ce

Mole ratioVCMC/(VCMC+VBTB)

00.10.20.30.40.50.6

0 0.2 0.4 0.6 0.8 1

Abso

rban

ce

Mole ratioVCMC/(VCMC+VBCG)

0

0.1

0.2

0.3

0.4

0.5

0 0.2 0.4 0.6 0.8 1

Abs

orba

nce

Mole ratio VCMC/(VCMC+VBCP)

0

0.2

0.4

0.6

0.8

1

0 5 10 15 20 25 30 35

Abso

rban

ce

Concentration of CMC, µg ml-1

0

0.2

0.4

0.6

0.8

1

0 5 10 15 20 25 30

Abso

rban

ce

Concentration of CMC, µg ml-1

130

Method C

Figure 4.2.4 Calibration curves

Table 4.2.1.Sensitivity and regression parameters

Parameter Method A Method B Method C

max, nm 420 415 425

Linear range, µg ml-1 2-35 1.6-28 1.6-28

Color stability, hrs 2 3 2.5

Molar absorptivity(ε), l mol-1 cm-1 1.5× 104 1.8 × 104 1.8 × 104

Sandell sensitivitya, µg cm-2 0.0383 0.0318 0.0329 Limit of detection (LOD), g ml-1 0.33 0.51 0.62 Limit of quantification (LOQ), g ml-1 0.99 1.55 1.89 Regression equation, Yb

Intercept (a) 0.0016 0.0063 0.0205 Slope (b) 0.0262 0.0307 0.0345 Standard deviation of a (Sa) 0.0326 0.0917 0.0923

Standard deviation of b (Sb) 0.0013 0.0042 0.0048 Regression coefficient (r) 0.9988 0.9997 0.9978

a Limit of determination as the weight in µg ml-1 of solution, which corresponds to an absorbance ofA = 0.001 measured in a cuvette of cross-sectional area 1 cm2 and l = 1 cm.b Y=a+bX, Where Y is the absorbance, X is concentration in µg ml-1. Precision and accuracy

The precision of the proposed methods was calculated in terms of

intermediate precision (intra-day and inter-day). Three different concentration of

CMC (within the working limits) were analyzed in seven replicates during the

same day (intra-day precision) and five consecutive days (inter-day precision).

The RSD (%) values of intra-day and inter-day studies showed that the precision

was good for all the three methods (Table 4.2.2). The accuracy of the methods

was evaluated as percentage relative error between the measured concentrations

0

0.2

0.4

0.6

0.8

1

0 5 10 15 20 25 30

Abso

rban

ce

Concentration of CMC, µg ml-1

131

and taken concentrations for CMC (Bias %). The results, compiled in Table 4.2.2,

show that the accuracy is good for all the three methods.

Table.4.2.2. Results of intra-day and inter-day accuracy and precision study

Method

CMC taken

µg ml-1

Intra-day accuracy and precision

(n=5)

Inter-day accuracy and precision

(n=5) CMC found

µg ml-1 %RE %RSD

CMC found

µg ml-1 %RE %RSD

A

10.0 10.20 1.78 1.11 10.25 1.65 1.18

20.0 20.31 1.58 2.53 20.38 1.72 2.48

30.0 30.35 1.19 0.91 30.46 1.59 1.41

B

8.0 7.86 1.66 1.87 7.92 2.06 1.76 16.0 16.07 0.47 1.45 16.04 1.47 2.05

24.0 24.27 1.14 0.63 24.35 1.84 1.23

C

8.0 8.08 1.08 1.41 8.13 1.38 1.38 16.0 15.89 0.63 1.28 15.78 1.43 1.42

24.0 23.52 1.95 1.74 23.67 1.90 1.66 RE- Relative error and RSD- Relative standard deviation

Selectivity

A systematic study was performed to determine the effect of matrix by

analyzing the placebo blank and synthetic mixture containing CMC. Placebo

blank and synthetic mixtures were prepared as described under Section 4.1.3.3.

The results obtained from placebo blank and synthetic mixture analyses revealed

that the inactive ingredients used in the preparation did not interfere in the assay

of active ingredient. The absorbance values obtained from the placebo blank

solution were almost equal to the absorbance of the blank which revealed no

interference from the adjuvants. To study the role of additives added to the

synthetic sample, 2 ml of the resulting solution prepared by using synthetic

mixture (50 µg ml-1 in CMC from method A and 40 µg ml-1 from method B and

method C) was assayed (n = 4). The percentage recoveries of 97.84 – 102.75 with

RSD (%) values in the range 1.09 – 2.43 demonstrated the accuracy as well as the

precision of the proposed methods and complement the findings of the placebo

blank analysis with respect to selectivity.

132

Robustness and ruggedness

The robustness of the methods was evaluated by making small incremental

changes in the volume of dye and contact time, and the effect of the changes was

studied on the absorbance of the ion-pair complex systems. The changes had

negligible influence on the results as revealed by small intermediate precision

values expressed as % RSD (≤ 1.82%). Method ruggedness was demonstrated by

having the analysis done by four analysts, and also by a single analyst performing

analysis on four different instruments in the same laboratory. Intermediate

precision values (%RSD) in both instances were in the range 1.64 – 3.27%

indicating acceptable ruggedness. The results are presented in Table 4.2.3.

Table 4.2.3. Results of robustness and ruggedness expressed as intermediate

precision (%RSD)

a Dye (BTB, BCG and BCP) volumes used were 0.9, 1.0 and 1.1 ml.

b Reaction time were 4.0, 5.0 and 6.0 min Application

The proposed methods were applied for the quantification of CMC in

commercial tablets. The results obtained were compared with those obtained using

official method [2].Statistical analysis of the results did not detect any significant

difference in the performance of the proposed method to the reference method

with respect to accuracy and precision as revealed by the Student’s t-value and

variance ratio F-value. The results of this study are given in Table 4.2.4

Method

CMC taken,

µg ml-1

Method robustness Method ruggedness Parameters altered

Dye, mla

RSD, % (n = 3)

Time, minb RSD,% (n=3)

Inter-analysists’ RSD, % (n = 4)

Inter-cuvettes’ RSD, % (n = 3)

A

10.0 1.13 1.28 1.46 2.86 20.0 1.19 1.54 0.92 3.27

30.0 1.26 1.31 0.84 1.64

B

8.0 1.34 1.82 0.76 2.66

16.0 1.28 1.12 1.26 3.25 24.0 1.23 1.64 1.01 3.12

8.0 1.26 1.42 1.56 2.83

C 16.0 1.21 1.17 0.84 2.46 24.0 1.17 1.29 1.16 3.14

133

Table 4.2.4. Results of analysis of tablets by the proposed methods and

comparison with the reference method

*mg/tablet in tablets .a Mean value of five determinations.

Recovery study

To further assess the accuracy of the proposed methods, recovery

experiment was performed by applying the standard-addition technique. The

recovery was assessed by determining the agreement between the measured

standard concentration and added known concentration to the sample. The test

was done by spiking the pre-analysed tablet powder with pure CMC at three

different levels (50, 100 and 150 % of the content present in the tablet powder

(taken) and the total was found by the proposed method. Each test was repeated

three times. From this test the percentage recovery values were found in the range

of 98.85 to 102.55 with standard deviation values from 0.62 – 2.51%. Closeness

of the results to 100% showed the fairly good accuracy of the method. These

results are shown in Table 4.2.5.

Tablet Brand nameb

Label claim*

Founda (Percent of label claim ±SD)

Reference method

Proposed methods A B C

Siphene-25

25

99.36±0.68

98.81±1.19 t =1.53 F=3.06

100.46±14 t =2.29 F =3.88

98.97±1.12 t =0.64 F =2.71

Siphene-100 100

100.92±0.72

99.60±1.37 t=2.76 F=3.62

100.24±1.2 t =1.75 F=3.16

100.76±1.3 t =0.82 F =2.52

134

Table 4.2.5. Results of recovery study by standard addition method

aMean value of three determinations.

Tablets

studied

Method A Method B Method C

CMC in

tablets,

µg ml-1

Pure

CMC

added,

µg ml-1

Total

found,

µg ml-1

Pure CMC

recovered*,

Percent±SD

CMC in

tablets,

µg ml-1

Pure

CMC

added,

µg ml-1

Total

found,

µg ml-1

Pure CMC

recovered*,

Percent±SD

CMC

in

tablets

µg ml-1

Pure

CMC

added,

µg ml-1

Total

found,

µg ml-1

Pure CMC

recovered*,

Percent±SD

Siphene

25

9.88

9.88

9.88

5

10

15

14.97

21.51

24.59

101.92±1.70

99.24±1.15

99.07±2.25

8.03

8.03

8.03

4

8

12

12.06

16.00

20.30

100.92±1.68

99.65±1.11

102.27±2.54

7.91

7.91

7.91

4

8

12

11.88

15.95

19.89

99.44±2.24

100.49±1.47

99.90±0.81

Siphene

100

9.96

9.96

9.96

5

10

15

14.94

21.63

24.76

99.77±1.25

100.47±0.62

98.85±0.75

8.01

8.01

8.01

4

8

12

12.11

16.09

20.10

102.55±2.51

101.02±1.11

100.76±0.50

8.06

8.06

8.06

4

8

12

12.12

16.18

19.94

101.69±1.72

101.58±1.50

99.01±1.14

135

Section 4.3

SIMPLE SPECTROPHOTOMETRIC ASSAY OF CLOMIPHENE CITRATE

IN PHARMACEUTICALS THROUGH CHARGE-TRANSFER

COMPLEXATION REACTION

4.3.2 INTRODUCTION

The chemistry and analytical utility of charge transfer complexation reaction

in spectrophotometric assay of organic compounds of pharmaceutical importance has

been reviewed in Section 2.3.1. The literature survey presented in Section 4.0.2.1

reveals a spectrophotometric method [11] based on charge transfer complexation

reaction, where DDQ is used as a chromogenic agent in chloroform medium. The

method involves the conversion of CMC to free base through base treatment followed

by extraction in to chloroform before reacting with DDQ in the same medium. This

looks tedious, prone to loss of analyte and seems less sensitive. By employing

dioxane-acetonitrole medium, the performance characteristics were greatly improved

by the author where extraction step is completely eliminated. Besides DDQ, p-

chloranilic acid (p-CAA) was also employed thus leading to two methods based on C-

T complex formation reactions, where CMC serves as n-donor and p-CAA in method

A and DDQ in method B as π-acceptors. The details are present in this Section, 4.3.

4.3.2 EXPERIMENTAL

4.3.2.1 Apparatus

The instrument used for absorbance measurements was the same as described

in Section 2.2.2.1.

4.3.2.2 Materials

1,4-Dioxane and acetonitrile (spectroscopic grade) were purchased from

Merck, Mumbai, India. All other chemicals used were of analytical reagent grade.

The pure CMC and its tablets used were the same as described in Section 4.1.

4.3.2.3 Reagents

p-Chloranilic Acid (0.1 %, w/v): The solution was prepared by dissolving 0.10 g of

p-chloranilic acid (Rolex lab reagents, India) in 100 ml of dioxane.

DDQ (0.1 %, w/v): The solution was prepared by dissolving 0.10 g of DDQ (Merck,

Mumbai, India) in 100 ml of 1,4-dioxane (Merck, Mumbai, India).

136

Standard CMC stock solution

For method A, a 200 µg ml-1 CMC stock solution was prepared by dissolving

20 mg of pure drug in acetonitrile in a 100 ml volumetric flask and the solution was

diluted to the mark with the same solvent, and the above 200 µg ml-1 CMC stock

solution was diluted with acetonitrile to get 100 µg ml-1 and used for the assay in

method B.

2.3.2.4 General procedures

Method A

Varying aliquots (0.2, 0.5, 1.0, 2.0, 3.0 and 4.0 ml) of CMC solution (200 µg ml-1)

were accurately measured into a series of 5 ml calibrated flasks by means of micro

burette. To each flask was added 1 ml of 0.1 % p-CAA, diluted to the mark with

acetonitrile and mixed well. Then, the absorbance was measured at 530 nm against

reagent blank treated similarly

Method B

Aliquots of 0.2, 0.5, 1.0, 2.0, 3.0 and 4.0 ml CMC standard solution (100 µg ml-1)

were transferred into a series of 5 ml calibrated flask. To each flask 1 ml of 0.1 %

DDQ solution was added, diluted to the mark with acetonitrile and mixed well. Then,

the absorbance was measured at 580 nm against reagent blank treated similarly.

A calibration graph was prepared by plotting the increasing absorbance values

versus concentration of CMC. The concentration of CMC was read from the

calibration graph or computed from the respective regression equation derived using

the Beer’s law data.

Procedure for tablets

An amount of tablet powder equivalent to 20 mg CMC was transferred into a

100 mL volumetric flask and about 60 ml of acetonitrile was added to the flask. The

content was shaken well for 20 min and diluted to the mark with the same solvent.

The resulting solution was filtered through Whatmann No 42 filter paper and used for

the assay by following the general procedure described for method A. This tablet

extract (200 µg ml-1) was diluted to 100 µg ml-1 with acetonitrile and suitable aliquot

was used for the assay in method B.

137

Procedure for the analysis of placebo blank and synthetic mixture

Placebo blank and synthetic mixtures were prepared as described in Section

4.1.2.4. A 20 mg of the placebo blank was accurately weighed and its solution was

prepared as described under ‘tablets’, and then subjected to analysis by following the

general procedures.

An amount of synthetic mixture equivalent containing 20 mg CMC was

accurately weighed and transferred into a 100 ml volumetric flask and the extract

equivalent to 200 µg ml-1 CMC was prepared as described under the general

procedure for tablets and used in method A. Required volume of the above extract

was diluted to 100 µg ml-1 with acetonitrile and used for method B by following the

general recommended procedure.

4.3.3 RESULTS AND DISCUSSION

π-acceptors like p-CAA and DDQ are known to yield radical ions via charge

transfer complexation reaction with a variety of n-donors including amines, iodide ion

and metallic salts [58-62]. The structural formula of CMC features an amino group;

therefore, attempts were made to determine CMC based on the formation of charge-

transfer complex with p-CA and DDQ as reagents.

Spectral characteristics and reaction pathway

CMC, a nitrogenous base acting as n-donor was made to react with two π-

acceptors, namely, p-CA and DDQ, to produce coloured charge transfer complexes in

1,4-dioxane-acetonitrile solvent system according to the following equation:

CMC + A CMC-A CMC+

+ A. -

C-T complex Radical anion In the method A, CMC reacts with the reagent and gives a red chromogen that

exhibits a strong absorption maximum at 530 nm in dioxane-acetonitrile medium

(Figure 4.3.1). This can be attributed to the formation of charge-transfer complex

between CMC and p-CAA followed by the formation of radical ions which probably

was due to the dissociation of the original (CMC-p-CAA) complex promoted by the

high ionizing power of the acetonitrile solvent [63].

In the second method, the interaction of CMC with DDQ in dioxane-

acetonitrile medium at room temperature gave a purple colored chromogen with

138

strong absorption maxima at 460, 540 and 580 nm due to the formation of the free

radical anion [64] and the wavelength 580 was selected for further studies because of

higher sample absorbance and lower blank absorbance readings (Figure 4.3.1).

Figure 4.3.1 Absorption spectra of: a. CMC-p-CAA C-T complex, b. blank (Method

A); c. CMC-DDQ C-T complex and d. blank. (Method B).

4.3.3.1 Method development

Optimum conditions were established by measuring the absorbance of C-T

complexes at 530 and 580 nm, for method A and method B, respectively, by varying

one parameter and keeping other parameters fixed.

Effect of reagent concentration

To establish optimum concentration of the reagents for the sensitive and rapid

formation of the CMC charge transfer complexes, the drug (CMC) was allowed to

react with different volumes of the reagents (0.5 – 2.5 ml of 0.1% p-CA and 0.5 - 3

ml of 0.1% DDQ). In both the cases, maximum and minimum absorbance values

were obtained for sample and blank, respectively, only when 1 ml of the reagent was

used. Therefore, 1 ml of reagent in a total volume of 5 ml was used throughout the

investigation.

Choice of solvent to dissolve drug and reagents

To dissolve CMC, acetonitrile was preferred to chloroform, dichloromethane,

acetone, 2-propanol, 1,2-dichloroethane, 1,4-dioxane, methanol and ethanol, because

as the complex formed in these solvents either had very low absorbance values or

precipitated upon dilution. Where as in the case of reagents, highly intense coloured

products were formed when 1,4-dioxane medium was maintained as solvent to

00.10.2

0.30.40.5

0.60.7

400 440 480 520 560 600 640 680 720

Abso

rban

ce

Wavelength, nm

a

b0

0.10.20.30.40.50.60.7

400 440 480 520 560 600 640 680 720

Abso

rban

ce

Wavelength, nm

c

d

139

dissolve p-CAA and DDQ. Therefore, acetonitrile and 1,4-dioxane were chosen as

solvents to dissolve CMC and the reagents, respectively.

Effect of reaction time and stability of the C-T complexes

The optimum reaction times were determined by measuring the absorbance of

the formed complex upon the addition of reagent solution to CMC solution at room

temperature. In both the methods the formation of C-T complex was complete within

5 min and the absorbance values of CMC-p-CAA and CMC-DDQ complexes were

stable for 2 h and 30 min, respectively.

Investigation of composition of C-T complexes

The composition of the C-T complexes with either p-CAA or DDQ was

evaluated by following the Job’s continuous variations method [57]. The experiments

were performed by preparing and mixing equimolar solutions of drug and reagent

(method A: 3.48 × 10-4 M CMC and p-CAA; method B: 1.67 × 10-4 M each) by

maintaining the total volume at 2.5 ml. The plots of the molar ratio between drug and

reagent versus the absorbance values were prepared (Figure 4.3.2a and 2b), and the

results revealed that the formation of C-T complex between drug and reagent

followed a 1:1 reaction stoichiometry. This finding was anticipated by the presence of

one basic electron donating center (nitrogen atom) in the CMC structure. Based on

this fact the following reaction pathway for the formation of C-T complex is proposed

and sown in Scheme 4.3.1.

(a) (b)

Figure 4.3.2 Job’s plots obtained for: (a) CMC-p-CAA C-T complex and (b)

CMC-DDQ C-T complex

0

0.1

0.2

0.3

0.4

0.5

0 0.2 0.4 0.6 0.8 1

Abso

rban

ce

Mole ratioVCMC/(VCMC+Vp-CA)

0

0.1

0.2

0.3

0.4

0.5

0 0.2 0.4 0.6 0.8 1

Abso

rban

ce

Mole ratioVCMC/(VCMC+VDDQ)

140

CMC+. + p-CAA-.

p-CAA C-T complex (1:1)

p-CAA radical anion measured at 530 nm

CMC+. DDQ-.

DDQ radical anion measured at 580 nm

CN

C N

O

O

Cl

Cl

DDQ

Cl O N

HO

O

OH

O OH

OH

O

Cl O N

HO

O

OH

O OH

OH

O

O

O

Cl

OHCl

HOCl O N

HO

O

OH

O OH

OH

O

O

O

Cl

OHCl

HO

Cl O N

HO

O

OH

O OH

OH

O

CMC

CMC

CN

C N

O

O

Cl

Cl

C-T complex (1:1) Scheme 4.3.1. Proposed reaction pathways for the formation of C-T complexes

between CMC and p-CAA/DDQ.

4.3.3.2 METHOD VALIDATION

The proposed methods were validated for linearity, sensitivity, precision, accuracy,

robustness, ruggedness, selectivity and recovery according to the International

Conference on Harmonization (ICH) [27] guidelines.

Linearity, sensitivity, limits of detection and quantification

Under optimum experimental conditions for CMC determination, the standard

calibration curves for CMC with p-CAA and DDQ were constructed by plotting

absorbance versus concentration (Fig. 4.3.3). A linear correlation between absorbance

at max and concentration values regression analysis of the Beer’s law data, optical

characteristics such as Beer’s law limits, molar absorptivity and Sandell sensitivity

values, limits of detection (LOD) and quantitation (LOQ) values of both the methods

are evaluated and they are presented in Table 4.3.1. The moderate values of ε and

Sandell sensitivity and LOD indicate the sensitivity of the proposed methods.

141

Method A Method B

Figure 4.3.3 Calibaration curves

Table 4.3.1.Sensitivity and regression parameters

Parameter Method A Method B

max, nm 530 580

Linear range, µg ml-1 8.0-160 4.0-80

Color stability, min 120 30

Molar absorptivity(ε), l mol-1 cm-1 3.4 × 103 7.5 × 103

Sandell sensitivity, µg cm-2 0.1714 0.0790

Limit of detection (LOD), g ml-1 2.90 1.23

Limit of quantification (LOQ), g ml-1 8.77 3.71

Regression equation

Intercept (a) 0.0059 0.0070

Slope (b) 0.0056 0.0132 Standard deviation of a (Sa) 0.0436 0.0577 Standard deviation of b (Sb) 0.0024 0.0053

Regression coefficient (r) 0.9998 0.9993 a Limit of determination as the weight in µg ml-1 of solution, which corresponds to an absorbance ofA = 0.001 measured in a cuvette of cross-sectional area 1 cm2 and l = 1 cm.b Y=a+bX, Where Y is the absorbance, X is concentration in µg ml-1. Precision and accuracy

The intra and inter-day accuracy and precision of the proposed methods were

evaluated. The results of this study are summarized in Table 4.3.2. The percentage

relative standard deviation (RSD, %) values were ≤ 0.94% (intra-day) and ≤ 2.32%

0

0.2

0.4

0.6

0.8

1

0 20 40 60 80 100 120140 160

Abso

rban

ce

Concentration of CMC, µg ml-1

0

0.2

0.4

0.6

0.8

1

1.2

0 20 40 60 80

Abso

rban

ce

Concentration of CMC, µg ml-1

142

(inter-day) indicating high precision of the methods. Accuracy was evaluated as

percentage relative error (RE, %) between the measured mean concentrations and

taken concentrations for CMC. The percentage relative error was calculated at each

concentration and these results are also presented in Table 4.3.2. Percent relative

error (RE, %) values of ≤ 1.86% demonstrates the high accuracy of the proposed

methods.

Table.4.3.2. Results of intra-day and inter-day accuracy and precision study

Method

CMC taken

µg ml-1

Intra-day accuracy and precision

(n=5)

Inter-day accuracy and precision

(n=5) CMC found

µg ml-1 %RE %RSD

CMC found

µg ml-1 %RE %RSD

A

40.0 40.26 0.65 0.94 40.44 0.99 1.42 80.0 80.82 1.02 0.74 80.97 1.86 1.78

120.0 121.26 1.05 0.51 121.52 1.72 1.64

B 20.0 19.73 1.08 0.72 19.89 1.33 1.94 40.0 40.98 1.45 0.37 41.06 1.85 1.64 60.0 60.46 0.76 0.68 60.78 0.94 2.32

RE- Relative error and RSD- Relative standard deviation

Selectivity

The results obtained from placebo blank and synthetic mixture analyses

revealed that the inactive ingredients used in the preparation did not interfere in the

assay of active ingredient. The absorbance values obtained from the placebo blank

solution were almost equal to the absorbance of the blank which revealed no

interference from the adjuvants. To study the role of additives added to the synthetic

sample, 2 ml of the resulting solution prepared using synthetic mixture (200 and 100

µg ml-1 in CMC from method A and method B) was assayed (n = 4). The percentage

recoveries of 98.63-102.38 with %RSD values in the range 1.08-1.95 demonstrated

the accuracy as well as the precision of the proposed method and complement the

findings of the placebo blank analysis with respect to selectivity.

143

Robustness and ruggedness

The robustness of the methods was evaluated by making small incremental

changes in the volume of reagent and contact time, and the effect of the changes was

studied on the absorbance of the complex systems. The changes had negligible

influence on the results as revealed by small intermediate precision values expressed

as RSD (≤ 1.36%). Method ruggedness was demonstrated by having the analysis done

by four analysts, and also by a single analyst performing analysis on four different

instruments in the same laboratory. Intermediate precision values (RSD, %) in both

instances were in the range 0.54-3.42% indicating acceptable ruggedness. The results

are presented in Table 4.3.2.

Table 4.3.2 Results of robustness and ruggedness expressed as intermediate precision

study (% RSD).

Method CMC taken,

µg ml-1

Robustness (%RSD)

Ruggedness

Inter-analysts (%RSD), (n=4)

Inter-instruments (%RSD),

(n=4)

Parameters altered* Volume Reaction

o of dye time

A 40.0 80.0 120.0

1.26 0.96 1.21 1.28 1.17 0.72

1.56 0.84 1.72

2.63 3.42 2.84

B 20.0 40.0 60.0

1.36 1.06 1.28 0.85 1.23 1.14

0.76 1.26 1.01

3.12 2.46 3.42

*The volumes of p-CAA or DDQ added were 1±0.2 ml; reaction time were 4,5 and 6 min.

Application to tablets

The proposed methods were successfully applied to the determination of CMC

in commercial tablets. The results obtained by the proposed methods were compared

to those of the reference method [2] by applying Student’s t-test for accuracy and F-

test for precision. The results (Table 4.3.4) show that the Student’s t- and F-values at

95 % confidence level are less than the theoretical values, which confirmed that there

is a good agreement between the results obtained by the proposed methods and the

reference method with respect to accuracy and precision.

144

Table 4.3.4. Results of analysis of tablets by the proposed methods and statistical

comparison of the results with the official method.

#Mean value of five determinations Recovery study

The accuracy and validity of the proposed methods were further ascertained by

performing recovery studies. Pre-analyzed tablet powder was spiked with pure CMC

at three concentration levels (50, 100 and 150 % of that in tablet powder) and the total

was found by the proposed methods. In both the cases, the added CMC recovery

percentage values ranged of 99.00-102.28 % with standard deviation of 0.62-2.71

(Table 4.3.5) indicating that the recovery was good, and that the co-formulated

substances did not interfere in the determination.

Tablets brand name

Labeled amount

(mg)

Found# (Percent of label claim ± SD) Official method

Method A Method B

Siphene-25

25

99.36±0.68

99.33±1.65 t = 0.53 F = 5.88

99.66±0.95 t =1.19 F =1.96

Siphene-100

100

100.92±0.72

99.52±1.52 t = 2.69 F =4.45

100.64±1.05 t = 1.14 F =2.23

145

Table 4.3.5. Results of recovery study via standard-addition method with tablets.

*Mean value of three determinations

Tablets

studied

Method A Method B

CMC

in

tablet,

µg ml-1

Pure

CMC

added,

µg ml-1

Total

found,

µg ml-1

Pure CMC

recovered

(Percent±SD*)

CMC

in tablet,

µg ml-1

Pure

CMC

added,

µg ml-1

Total

found,

µg ml-1

Pure CMC

recovered

(Percent±SD*)

Siphene -

25

59.75

59.75

59.75

30.0

60.0

90.0

76.54

101.02

127.35

102.03±1.28

101.61±0.62

100.03±0.90

29.90

29.90

29.90

15.0

30.0

45.0

45.16

59.63

74.57

101.79±2.64

99.10±2.07

99.28±1.24

Siphene-

100

59.60

59.60

59.60

30.0

60.0

90.0

99.29

101.36

124.40

101.41±0.83

102.28±1.30

99.60±1.57

30.2

30.2

30.2

15.0

30.0

45.0

45.52

59.90

75.78

102.15±2.71

99.00±1.04

101.30±1.55

146

Section 4.4

A STABILITY -INDICATING UPLC METHOD FOR THE

DETERMINATION OF CLOMIPHENE CITRATE IN PHARMACEUTICALS

4.4.1 INTRODUCTION

The utility and importance of UPLC was discussed in Section 3.3.1. The

reported chromatographic methods as seen from the literature survey presented in

Section 4.0. deal with either separation of the E and Z isomers, determination in body

fluids or some interactions, and no assay method has ever been reported

To the best of the author’s knowledge, no UPLC method has been ever

reported for the determination of CMC in pharmaceuticals, Thus, a need for a rapid,

precise, accurate and validated stability-indicating UPLC method for the

determination of CMC in bulk and tablets was felt. This was accomplished with a

Waters Acquity UPLC system and Acquity BEH column (C18, 100mm, 2.1mm and

1.7 µm). The stability indicating power of the method was established by comparing

the chromatograms obtained under optimized conditions before forced degradation

with those after degradation via acidic, basic, hydrolytic, oxidative, thermal and

photolytic stress conditions. The optimization parameters and the validation results in

detail are presented in this Section (4.4).

4.4.2 EXPERIMENTAL

4.4.2.1 Materials

All the reagents used were of analytical grade. Doubly distilled water was

used throughout the investigation. Pure CMC and tablets used were the same as

described in Section 4.1. HPLC grade acetonitrile was purchased from Merck India

.Pvt. Ltd, Mumbai, India.

4.4.2.2 Reagents and solutions

Hydrochloric acid (2M) was prepared by appropriate dilution of the concentrated acid

(Merck, Mumbai, India, Sp. gr. 1.18) with water. Sodium hydroxide (2M) was

prepared by dissolving accurately weighed sodium hydroxide pellets (Merck,

Mumbai, India) in water. H2O2 (5%) was prepared as described in Section 3.3.2.

147

Preparation of stock solution

A 800 µg ml-1 CMC stock standard solution was prepared by dissolving

accurately weighed 80 mg of pure CMC in mobile phase and diluted to mark in a 100

ml standard flask with the mobile phase.

4.4.2.3 Mobile phase preparation

Dissolved 1.0 g of potassium dihydrogenorthophosphate (Merck, Mumbai,

India) in 1000 ml of water and the pH was adjusted to 3.5 using NaOH and

triethylamine (Merck, Mumbai, India). A 650 ml portion of this resulting buffer was

mixed with 350 ml of acetonitrile, shaken well and filtered using 0.22 µm Nylon

membrane filter. This solution was also used as diluent in all subsequent preparations

of the sample.

3.4.2.4 Chromatographic conditions and equipments

Chromatographic analysis was performed on a Waters Acquity UPLC™

system (Waters, Manchester, UK) using an Acquity BEH column (C-18 100 mm, 2.1

mm and 1.7 µm; Waters, Manchester, UK) equipped with binary solvent delivery

pump, auto sampler and tunable UV (TUV) detector. The column oven temperature

was maintained at 35 °C and the autosampler was maintained at ambient temperature.

Gradient mobile phase flow was carried out throughout the run. Total cycle time was

5 min with a flow rate of 0.2 ml min-1 and an injection volume of 2 µl using partial

loop mode. The output signal was monitored and processed using Empower-2

software.

4.4.2.5 Instrumental parameters

The isocratic flow rate of mobile phase was maintained at 0.2 ml min-1. The

column temperature was adjusted to 35 °C. The injection volume was 2.0 µl. The

sample run was monitored at 230 nm and the run time was 5.0 min. The retention

time of the sample was observed at about 2.2 min.

4.4.2.4 Stress study

All stress decomposition studies were performed at an initial drug

concentration of 80 µg ml-1 in mobile phase. Acid hydrolysis was performed in 2 M

HCl at 80 °C for 2 h. The study in alkaline condition was carried out in 2 M NaOH at

80 °C for 2 h. Oxidative studies were carried out at 80 °C in 5% hydrogen peroxide

148

for 2 h. For photolytic degradation studies, pure drug in solid state was exposed to 1.2

million flux hours in a photo stability chamber. Additionally, the drug powder was

exposed to dry heat at 105 °C for 3 h. Samples were withdrawn at appropriate time,

cooled and neutralized by adding base or acid and subjected to UPLC analysis after

suitable dilution.

4.4.3 General procedures

Procedure for preparation of calibration curve

Working standard solutions containing 0.5-200 µg ml-1 CMC were prepared

by serial dilutions of aliquots of the stock solution. Aliquots of 2 µl were injected (six

injections) and eluted with the mobile phase under the reported chromatographic

conditions. The average peak area versus the concentration of CMC in µg ml-1 was

plotted. Alternatively, the regression equation was derived using mean peak area-

concentration data and the concentration of the unknown was computed from the

regression equation.

Preparation of tablet extracts and assay procedure

Ten Siphene (25 and 100 mg CMC) tablets were accurately weighed and

ground into a fine powder. Powder equivalent to 8 mg CMC was transferred into a

100 ml volumetric flask and 60 ml of the mobile phase was added. The mixture was

sonicated for 20 min to achieve complete dissolution of CMC, and the content was

then diluted to volume with the mobile phase to yield a concentration of 80 µg ml-1

CMC, and filtered through a 0.22 µm nylon membrane filter. The tablet extract was

injected to the UPLC column.

Procedure for method validation

Accuracy and Precision

Six injections, of three different concentrations (60, 80 and 100 µg ml-1), were

given on the same day and the values of relative standard deviation (%RSD) were

calculated to determine intra-day precision. These studies were also repeated on

different days to determine inter-day precision. The same results were also used to

assay the accuracy of the method also.

149

Limits of detection (LOD) and quantification (LOQ)

Signal to noise (S/N) ratio method was adopted to obtain the limit of

quantification (LOQ) and limit of detection (LOD). Series of dilutions of the CMC

stock solution was made to attain LOQ and LOD in acceptable values.

Linearity

Seven-point calibration curves were obtained in a concentration range from

0.5 to 200 µg ml−1 (0.5, 50, 100, 150 and 200 µg ml-1 levels) for CMC; three

independent determinations were performed at each concentration.

Robustness and Ruggedness

To determine the robustness of the method, experimental conditions were

deliberately changed. The flow rate of the mobile phase (0.2±0.05 ml min-1), column

oven temperature (35±5 ºC), mobile phase composition (60:40, 65:35 and 70:30

buffer: solvent mixture v/v) and detection wavelength (230±1 nm) were the varied

parameters. In each case, the RSD (%) values were calculated for the obtained peak

area and retention time. The number of theoretical plates and tailing factors were

compared with those obtained under the optimized conditions. Three different

columns of same dimensions were used for the analyses. The study was performed on

the same day and on three different days by three different analysts for three different

concentrations of CMC (triplicate injections). The area obtained from each

concentration was compared with that obtained under optimized conditions. The

percent relative standard deviation values were evaluated for each concentration.

Solution stability and mobile phase stability

Stability of CMC solution was studied by injecting the sample into the

chromatographic system at different time intervals. The peak area was recorded in the

time intervals of 0, 12 and 24 hrs and the RSD values were calculated. Freshly

prepared solution was injected at the same time intervals for mobile phase stability (0,

12 and 24 hours) and RSD values of the peak areas were calculated.

4.4.3 RESULTS AND DISCUSSION

4.4.3.1 Method development

Different chromatographic conditions were experimented to achieve better

efficiency of the chromatographic system. Parameters such as mobile phase

150

composition, wavelength of detection, column, column temperature, pH of mobile

phase and diluents were optimized. Several proportions of buffer, and solvents (water,

methanol and acetonitrile) were evaluated in-order to obtain suitable composition of

the mobile phase. Choice of retention time, tailing, theoretical plates and run time

were the major tasks while developing the method. Acquity BEH C18, 50 mm × 2.1

mm, 1.7 µm column used for the elution, but the peak eluted before 1.5 minutes with

a tailing factor of 2. The following gradient conditions were experimented; the cycle

time was set at 5 min, 10 min, 15 min or 20 min while the flow rate was set at either

0.2 ml min-1 or 0.6 ml min-1. Except for the 5 min cycle time, all gradients began with

100% buffer for 0.5 min and maintained for 1 min at the end of each cycle for

equilibration. For a cycle time of 5-min , conditions started with 100% buffer for 0.5

min, then proceeded with a linear gradient to 100% acetonitrile for 3 min, then

returned to initial conditions and maintained upto 5 min. The gradient method was

successful in giving good peak shape of drug. The effect of different elution gradients

was assessed under either linear (described above), curve or step gradient which was

controlled by the Waters Empower-2 software. At 65: 35 ratio of the mobile phase in

the linear gradient program, a perfect peak was eluted. Thus the mobile phase ratio

was fixed at 65:35 (buffer: solvent) in an isocratic mobile phase flow rate. The typical

chromatograms obtained for blank and pure CMC in final optimized UPLC

conditions are depicted in Figure 4.4.1.

(a)

151

(b)

Figure 4.4.1 Typical chromatograms obtained under optimized conditions for: (a) 80

µg ml-1 CMC and (b) blank

Stability study (Degradation study)

All forced degradation studies were performed at 80 µg ml-1 concentration

level. The observation was made based on the peak area of the respective sample.

CMC was found to be more stable under photolytic (1.2 million flux hours), thermal

(105 0C for 3 hours) in solid state, and hydrolytic (aqueous, 80 0C for 2 hours) stress

conditions. The drug was found to be slightly sensitive to acid (1%) and sensitive to

alkaline and oxidative stress conditions resulting in the decomposition to the extent of

15.2 and 44.5 %, respectively. The chromatograms obtained for CMC after subjecting

to degradation are presented in Figure 4.4.2.

(a)

152

(b)

(c)

(d)

153

(e)

(f)

Figure 4.4.2 Chromatograms obtained for CMC after subjecting to stress studies by: (a) acid degradation, (b) base degradation, (c) photo degradation, (d) thermal degradation (e )oxidative degradation and (f) control.

4.4.3.2 Method validation

Analytical parameters

A linear correlation was obtained between the peak area and the concentration

in the range of 0.5 – 200 µg ml-1 CMC from which the linear regression equation was

computed and found to be:

y = 30,620x + 19,330, r² = 0.9999

where y is the mean peak area, x is the concentration of CMC in µg ml-1 and r is the

correlation coefficient. The LOD and LOQ values, slope (m), y-intercept (a) and their

standard deviations are evaluated and presented in Table 4.4.1. These results confirm

154

the linear relation between concentration of CMC and the peak areas as well as the

sensitivity of the method.

Table 4.4.1 Linearity and regression parameters with precision data

Parameter Value Linear range, µg ml-1 0.5 -200 Limits of quantification, (LOQ), µg ml-1 0.5 Limits of detection, (LOD), µg ml-1 0.1 Regression equation Slope (b) 30620.1 Intercept (a) 19330.0 Correlation coefficient (r) 0.9999

Accuracy and precision

The percent relative error which is an indicator of accuracy is ≤1.0% and is

indicative of high accuracy. The calculated percent relative standard deviation (RSD,

%) can be considered to be satisfactory. The peak area based and retention time based

RSD values were <1%. The results obtained for the evaluation of accuracy and

precision of the method are compiled in Tables 4.4.2 and 4.4.3.

Table 4.4.2 Results of accuracy study (n=5)

Table 4.4.3 Results of precision study (n=5)

Concentration of CMC injected, µg ml-1

Intra-day Inter-day

Concentration of CMC found, µg ml-1

RE (%)

Concentration of CMC found, µg ml-1

RE (%)

60.0 60.32 0.64 60.49 0.98

80.0 79.58 0.42 88.97 0.88 100.0 99.44 0.37 99.12 0.85

Concentration injected, µg ml1

Intra-day precision Inter-day precision Mean area± SD RSDa

(%) RSDb (%)

Mean area±SD RSDa (%)

RSDb (%)

60.0 1676974±9012 0.29 0.38 1689372±9012 0.42 0.29 80.0 2235731±10694 0.84 0.26 2198192±10694 0.76 0.17 100.0 2794663±9715 0.65 0.41 2804321±9715 0.57 0.68 a Relative standard deviation based on peak area; bRelative standard deviation based on retention time.

155

Selectivity

The chromatogram of the placebo blank did not yield any peak. The

chromatogram of the synthetic mixture solution equivalent to100 µg ml-1 CMC

yielded a chromatogram containing a peak with an area equal to that of pure drug

solution of the same concentration (100 µg ml-1). These studies indicate that the

method is highly selective.

Robustness and ruggedness

The robustness of an analytical procedure is a measure of its capacity to

remain unaffected by small, but deliberate variations in method parameters, and

provides an indication of its reliability during normal usage. At the deliberate varied

chromatographic conditions (flow rate, temperature, and mobile phase composition),

the analyte peak RSD (%), tailing factor and theoretical plates remained closer to the

actual values. The RSD values ranged from 0.1 to 1.3% resumes the robustness of the

proposed method. In method ruggedness, different columns (same lot), on different

days were used and analyses were performed by different analysts. The results are

summarized in Tables 4.4.4 and 4.4.5.

Solution stability and mobile phase stability

At the specified time interval, RSD (%) for the peak area obtained from drug

solution stability and mobile phase stability were within 1%. This shows no

significant change in the elution of the peak and its system suitability criteria (RSD,

tailing factor, theoretical plates). The results also confirmed that the standard solution

of drug and mobile phase were stable for at least for 24 hours during the assay

performance.

156

Table 4.4.4 Results of method robustness study expressed as intermediate precision (%RSD)

*Mean value of three determinations (for 80 µg ml-1)

Table 3.3.5 Results of method ruggedness study expressed as intermediate precision (%RSD)

Variable Mean peak area ±SD* RSD, % Mean

Rt ± SD* RSD,

%

Mean theoretical plates ± SD

RSD, %

Mean tailing

factor±SD* RSD, %

Analyte (n=3) 2291157±9714 0.42 2.186±0.02 0.91 2216±28.63 1.29 1.44±0.007 0.48

Column (n=3) 2102015±10318 0.49 2.209±0.01 0.45 2392±21.33 0.89 1.42±0.006 0.42

*Mean value of three determinations (for 80 µg ml-1)

Condition Modification Mean peak area±SD* RSD,% Mean Rt± SD* RSD,% Mean theoretical

plates ±SD*

Mean tailing factor ± SD*

RSD,%

30oC 2281384±9826 0.43 2.188±0.016 0.73 2136±24.65 1.42±0.02 1.41 Temperature 35oC 2268637±10081 0.44 2.205±0.008 0.36 2093±32.36 1.43±0.01 0.69

40oC 2237163±11307 0.50 2.179±0.009 0.41 1962±43.56 1.45±0.01 0.68

Mobile phase Composition

60:40 2137164±10215 0.47 2.199±0.006 0.27 2237±19.36 1.44±0.009 0.62 65:35 2134782±9237 0.43 2.262±0.006 0.26 2189±22.56 1.45±0.01 0.68

70:30 2283672±9886 0.42 2.187±0.008 0.40 2078±36.97 1.47±0.015 1.02 0.15 2273871±8820 0.38 2.208±0.005 0.18 2256±24.16 1.42±0.02 1.40

Flow rate 0.20 2162920±8617 0.39 2.216±0.007 0.31 2151±28.84 1.45±0.02 1.36 0.25 2216519±9953 0.44 2.193±0.005 0.22 2038±32.16 1.47±0.01 0.68 229nm 2083672±11638 0.55 2.221±0.008 0.36 2156±25.16 1.44±0.02 1.38

Wavelength 230nm 2128654±10636 0.49 2.185±0.005 0.22 2298±31.32 1.43±0.009 0.62 231nm 2198732±9945 0.45 2.198±0.006 0.27 2356±28.58 1.41±0.01 0.70

157

Analysis of tablets

The described method was successfully applied to the determination of CMC

in its pharmaceutical tablet formulation. The results obtained (Table 4.4.6) were

statistically compared with those of reference method [2]. The results obtained by the

proposed methods agreed well with those of reference method and with the label

claim. The results were also compared statistically by a Student’s t-test for accuracy

and by a variance F-test for precision at 95 % confidence level as summarized in

Table 4.4.6 which reveals no significant difference between the proposed method and

the reference method with respect to accuracy and precision.

Table 4.4.6. Results of analysis of tablets by the proposed method and statistical comparison of the results with the official method.

#Mean value of five determinations

Recovery study

A standard addition procedure was followed to further evaluate the accuracy

of the method. The solutions were prepared by spiking pure drug into a pre-analyzed

tablet powder at three different levels and injected to chromatographic column. The

recovery of the known amount of added analyte was computed. The percentage

recovery of CMC from pharmaceutical dosage forms ranged from 99.07% - 101.32%.

The results given in Table 4.4.7 reveal good accuracy of the proposed method.

Tablet name Label

claim, mg

Found# (Percent of label claim ± SD) Official method

Proposed method

Siphene-25

25

99.36±0.68

99.43±1.65 t = 0.63 F = 3.88

Siphene-100

100

100.92±0.72

99.82±1.52 t = 1.29 F =4.45

158

Table 4.4.7 Results of recovery study by standard addition method

#Mean value of three determinations

Tablet studied

CMC in tablet,

µg ml-1

Pure CMC added, µg ml-1

Total found, µg ml-1

Pure CMC recovered* (%CMC±SD)

Siphene 25 49.71 49.71 49.71

25.0 50.0 75.0

75.05 100.51 124.38

101.32±1.28 100.82±0.85 99.69±0.97

Siphene 100 49.91 49.91 49.91

25.0 50.0 75.0

74.21 100.39 123.99

99.07±0.80 100.48±0.47 99.27±0.64

159

Section 4.5

SUMMARY AND CONCLUSIONS - Assessment of the methods

To sum up, two UV- and five visible spectrophotometric and one UPLC

methods were developed and validated for the determination of clomifene citrate in

bulk drug and in tablets. The UV-spectrophotometric and UPLC technique were

additionally used to evaluate the behavior of the drug towards several stress

conditions such as acid and base hydrolysis, peroxide oxidation, light and heat.

The performance characteristics of the proposed spectrophotometric methods

in comparison with the existing/reported methods are presented in Table 4.5.1. The

UV- spectrophotometric methods are simple and cost-effective in terms of the media

employed (HCl and H2SO4). Wide linear dynamic ranges (2-60 µg ml-1), high

sensitivity (=1×104) and low LOQ values (< 3 µg ml-1) are the features of the

proposed UV-spectrophotometric methods.

160

Table 4.5.1 Comparison of performance characteristics of proposed spectrophotometric methods with the existing methods Sl. No

Reagent/s used Methodology max (nm)

Linear range (µg ml-1) ( in l mol-1 cm-1)

LOQ (µg ml-1)

Remarks Ref

1 a)Methyl orange Measurement of chloroform extractable ion-pair complex

420 - Time-consuming extraction step is involved

9

b)Citricacid-acetic anhydride

Measurement of green

colored complex

605 - -

2 a)Iodine

b)Ammoniummolybdate

c)Phosphomolybdic acid

Measurement of molecular complex formed

Measurement of molecular

complex formed

Measurement of molecular complex formed

- 2-24

- Multistep reaction, time consuming, involves

precipitation, filtration steps.

10

3 DDQ Measurement of chloroform extractable

C-T complex

460 10-70 (4.61×103)

- The method involves conversion of the drug to base, extraction of the complex in to CHCl3 before measurement

11

4 Ammonium reineckate Measurement of ion-associate complex after

precipitation, washing and dissolution in acetone

509 200-1800 (2.6×102)

- Less sensitive and time consuming. is multi-step method

12

5

a) 0.1 M H2So4

Absorbance measured in either 0.1 M H2SO4 or 0.1 M HCl

290 2-60 1.0 × 104

2.42 Simple, sensitive, easy to perform

Present work

b) 0.1 M HCl

289 2-60 (1.0 ×104)

2.84

161

6 a) Bromothymol blue

Measurement of ion-pair complex formed in dichloromethane without extraction

420 2-35 (1.5× 104)

0.99 No extraction step, simple and sensitive with wide linear dynamic ranges

Present work

b) Bromocresol green 415 1.6-28 (1.8 × 104)

1.55

c) Bromocresol purple 425 1.6-28 (1.8 × 104)

1.89

7. a)p-CAA Measurement of C-T complex in acetonitrile-dioxane medium

530 8-160 (3.4 × 103)

8.77 Free from extraction step, sensitive with wide dynamic linear ranges, precise and accurate

Present work

c) DDQ 580 4-80 (7.5 × 103)

3.71

p-CAA- p-chloranilic acid, DDQ-2,3-dichloro 5,6-dicyano, p-benzoquinone.

The published visible spectrophotometric method [9] employing methyl orange as an ion-pair agent requires manipulation of several

experimental variables such as pH, aqueous-organic phase ratio, extraction time, equilibration time besides the laborious extraction

step. The C-T complex method [11] using DDQ requires the conversion of the drug to base form followed by extraction in to CHCl3

before absorbance measurement. The ammonium reineckate method [12] is a multi step process involving precipitation, filtration, and

dissolution of the precipitate in acetone before measuring the absorbance of the complex, thus leading to inaccuracy and imprecision.

These drawbacks are totally absent in the methods developed by the author. Both ion-pair and C-T complex methods are the simplest

in terms of ease of operation since they involve simple mixing of the drug and reagent solutions in appropriate solvents and

measurement of the absorbance. The absence of the manipulation of the experimental variables unlike in methods [8-12] renders the

proposed methods highly accurate and precise. Of the proposed five visible spectrophotometric (Table 4.5.1, Sl. No. 6 and 7),

162

procedures based on ion-pair reaction seem sensitive ( ˜ 2×104) compared those

based C-T complexation ( < 104). All the five methods are applicable over wide

linear dynamic ranges and found to be unaffected by the inactive ingredients added to

tablets as shown by the results of tablet analysis apart from placebo blank and

synthetic mixture analyses. The only limitation of these methods is the use of organic

solvent as the reaction medium. However, the use has been scaled down to the barest

minimum. With the exception of USP [2] and BP [8] methods, no other

chromatographic method is currently available for the clomifene citrate. The reported

chromatographic methods [13-21] deal with either separation of isomers or

determination of the drug in divergent matrices. The proposed UPLC method fills this

void since it essentially concerns with the assay of drug in pharmaceuticals and more

importantly the method is stability-indicating. The method has a wide linear dynamic

range (0.5 -200 µg ml-1), small LOD and LOQ values (0.1 and 0.5 µg ml-1) besides

being robust and rugged. The method looks highly specific as seen from the absence

of any peak from the placebo blank and any additional peak from synthetic

mixture/tablet chromatograms. High accuracy and precision are the other hallmarks

of the developed UPLC method.

163

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