CHAPTER II

TITRIMETRIC AND SPECTROPHOTOMETRIC ASSAY OF

MYCOPHENOLATE MOFETIL

16

Section 2.0

DRUG PROFILE AND LITERATURE SURVEY

2.0.1 DRUG PROFILE

Mycophenolate mofetil (MPM) is chemically known as 2-morpholinoethyl

(E)-6-(1,3-dihydro-4-hydroxy-6-methoxy-7-methyl-3-oxo-5-isobenzofuranyl)-4-

methyl-4-hexenoate. Its empirical formula is C23H31NO7 and molecular weight

433.50 g mol-1. MPM has the following chemical structure:

O

HO

O

OO

ON

O

Physically, MPM is a white crystalline powder. It is slightly soluble in

water; the solubility increases in acidic medium. It is freely soluble in acetone,

acetonitrile, acetic acid, hydrochloric acid, sulphuric acid, methanol, and sparingly

soluble in ethanol.

MPM is a new immunosuppressive drug [1]. MPM is the pro-drug of

mycophenolic acid (MPA), a medication used to treat psoriasis in the 1970s until

side effects and the concern of carcinogenesis led to its discontinuation [2].

Currently, MPM is indicated for the prevention of organ rejection in transplant

patients. MPM has recently been added to therapeutic regimens for skin disorders

[3]. MPA is a fivefold more potent inhibitor of the type II isoform of inosine

monophosphate dehydrogenase (IMPDH), which is expressed in activated

lymphocytes, than of the type I isoform of IMPDH, which is expressed in most

cell types. MPA has, therefore, a more potent cytostatic effect on lymphocytes

than on other cell types. This is the principal mechanism by which MPA exerts

immunosuppressive effects [4].

MPM has official monograph in British Pharmacopoeia [5]. In the

procedure of this standard monograph, MPM has been assayed potentiometrically

using 0.1 M perchloric acid in anhydrous acetic acid medium.

17

2.0.2 LITERATURE SURVEY OF ANALYTICAL METHODS FOR

MYCOPHENOLATE MOFETIL

2.0.2.1 Titrimetric and UV spectrophotometric methods

Other than the official method [5], no titrimetric procedures are found in

the literature for the determination of MPM either in its pure form or its dosage

form. However, two UV-spectrophotometric methods [6], in which the absorbance

of the drug solution either in 0.1 M HCl or in acetate buffer of pH 4.9 was

measured at 250 nm, were found in the literature.

2.0.2.2 Visible spectrophotometric methods

Although spectrophotometric methods in the visible region are the

instrumental methods of choice commonly used in laboratories, no

spectrophotometric method has ever been reported so far for the determination of

MPM either in bulk drug or in dosage form.

2.0.2.3 Chromatographic methods

High performance liquid chromatography (HPLC), [7-9], liquid

chromatography-mass spectrometry [10] and micellar electrokinetic capillary

chromatography [11] have been reported for the determination of MPM in

biological materials.

A liquid chromatographic method for the simultaneous determination of

MPM and its degradation product, mycophenolic acid (MPA) in dosage form [12]

is found in the literature. An HPLC method [13] was utilized for determining

MPM in capsules.

From the literature survey presented in the foregoing paragraphs, it is clear

that the only titrimetric method [5] is suitable for determining MPM at macro level

and cannot be applied at low levels as in a single tablet whenever required for checking

the content uniformity in tablets. Spectrophotometry is considered as the most convenient

analytical technique in pharmaceutical analysis because of its inherent simplicity and

availability in most quality control and clinical laboratories. But, except two UV-

spectrophotometric methods [6], no visible spectrophotometric method has ever

been reported for MPM. Considering the importance of both titrimetry and

spectrophotometry in pharmaceutical analysis, the author has applied these

techniques for the assay of MPM both in bulk drug and in dosage form.

The details concerning the method development and validation are

compiled in this Chapter.

18

Section 2.1

SPECTROPHOTOMETRIC ASSAY OF MYCOPHENOLATE

MOFETIL IN PHARMACEUTICALS USING CERIUM(IV) AND P-

DIMETHYLAMINOBENZALDEHYDE

2.1.1 INTRODUCTION

The ceric ion is a strong oxidizing agent, especially under acidic

conditions. When ceric compounds are reduced, so-called cerous compounds are

formed. The reaction taking place is:

Ce4+ + e− → Ce3+

Its powerful oxidizing property led its application in titrimetry in the late

19th century [14]. Nearly 70 years later, Willard and Young started to work

systematically using cerium(IV) [15]. Furman [16-18], Atanasiu [19] and their

collaborators described various potentiometric methods using cerium(IV) sulphate

as the oxidimetric titrant. As a result of this and later investigations, cerium(IV)

solutions have assumed considerable importance in oxidimetry. A comprehensive

review of the subject has been given by Young [20].

The oxidation potential of the ceric-cerium system depends on the acidity

(it hydrolyses to form ceric hydroxide if the solution is not acidic) and particularly

on the kinds of anions present; the value is 1.44 V in 1 N H2SO4 and 1.70 in

perchloric acid. In hydrochloric acid, elemental chlorine is formed, albeit slowly.

Therefore, it is always preferable to prepare its solution either in H2SO4 or

perchloric acid.

Cerium(IV) is widely used as oxidimetric reagent for the titrimetric and

spectrophotometric determination of numerous inorganic and organic substances

[21-24]. Titrimetric procedures have been employed for substances such as purine

derivatives [25], nitrofuron and pyrimidine derivative [26], papaverine

hydrochloride [27], ephedrine [28], salicylic acid [29], propranolol [30], atenolol

[31], ciprofloxacin [32], pantoprazole [33], isoxuprine [34] and olanzapine [35]

using cerium(IV) as oxidizing agent.

Apart from a variety of pharmaceutical substances which have been

determined by direct spectrophotometry using cerium(IV) as the oxidimetric

reagent, a few substances such as propranolol [30], atenolol [31], methylthiouracil

[36], antiamoebics and anthelmentics [37] and diuretics [38] have also been

determined by indirect spectrophotometric procedure based on different reaction

19

schemes. Very recently cerium(IV) was used for the determination of

oxcarbazepine by indirect spectrophotometry [39-41].

The literature survey presented in Section 2.0.2 reveals that cerium(IV)

has not used before for the assay of MPM. The author has made an attempt in this

direction and succeeded in developing a visible spectrophotometric method based

on the oxidation of drug with cerium(IV) in perchloric acid medium and

subsequent measurement of the excess cerium(IV) by its reaction with p-

dimethylaminobenzaldehyde to give a coloured product measurable at 460 nm.

The details are presented in this Section 2.1.

2.1.2 EXPERIMENTAL

2.1.2.1 Apparatus

A Systronics model 106 digital spectrophotometer (Systronics Ltd,

Ahmedabad, India) with 1 cm path length matched quartz cells was used to record

the absorbance values.

2.1.2.2 Materials

All chemicals used were of analytical reagent grade. Distilled water was

used throughout the investigation. Pharmaceutical grade MPM was procured from

Apotex Research Pvt Ltd, Bangalore, India, as a gift, and was used as received.

The purity of MPM was certified as 99.5%. CellCept 500 (Roche S.P.A., Italy)

(containing 500 mg MPM/tablet) tablets were obtained from the commercial

sources.

2.1.2.3 Reagents and solutions

Perchloric acid (HClO4, 4M): Prepared by diluting appropriate volume of

commercial acid (70%; Merck, Mumbai, India) with water.

Sulphuric acid (H2SO4, 1 M & 0.5 M): A 1 M acid was prepared by appropriate

dilution of concentrated acid (98%; Sp.gr., 1.84 Merck, Mumbai, India) with

water. This was diluted to 0.5 M with water, and used for the preparation of

cerium(IV) solution.

Cerium(IV) sulphate solution [Ce(IV), 300 µg ml-1]: A 0.025 M solution was

prepared by dissolving an accurately weighed quantity of ceric sulphate

[Ce(SO4)2.4H2O; assay 99%-from Loba Chemie Ltd, Mumbai, India] in 0.5 M

H2SO4 with the aid of heat. The solution was cooled to room temperature, and

filtered using glass wool. This solution after standardization [42] was diluted with

4 M HClO4 to get a working concentration of 300 µg ml-1 in Ce(IV) ion.

20

p-Dimethylaminobenzaldehyde (p-DMAB, 0.5%): An accurately weighed 1.25

g of p-DMAB (Merck, Mumbai, India) was transferred to a 250 ml volumetric

flask, dissolved in 4 M HClO4 and the volume was made upto the mark with the

same solvent.

Standard MPM solution

A 500 µg ml-1 stock MPM solution was prepared by dissolving an

accurately weighed 50 mg of pure drug in 4 M perchloric acid and the volume was

brought to 100 ml with the same solvent in a volumetric flask. The stock solution

was diluted 10 fold with the same solvent to get a working concentration of 50 µg

ml-1 MPM.

2.1.2.4 General procedures

Calibration curve

Different aliquots (0.25 – 6.0 ml) of standard 50 µg ml-1 MPM solution

were transferred into a series of 10 ml volumetric flasks using a microburette and

the total volume in all the flasks was adjusted to 6 ml by adding 4 M HClO4. To

each flask, 1 ml of 300 µg ml-1 Ce4+ solution was added, and the content was

mixed well and kept aside for 10 min at room temperature. Finally, 1 ml of 0.5%

p-DMAB was added to each flask and the volume was made up to mark with 4 M

HClO4. After 15 min, the absorbance of the coloured product was measured at 460

nm against water. A standard graph was prepared by plotting absorbance against

concentration and the unknown concentration was read from the graph or

computed from the regression equation derived using Beer’s law data.

Procedure for tablets

Ten CellCept 500 tablets were weighed and pulverized. A quantity of

tablet powder containing 5 mg of MPM was transferred into a 100 ml volumetric

flask. The content was shaken well with about 70 ml of 4 M HClO4 for 20 min.

The mixture was diluted to the mark with the same solvent and filtered using

Whatman No 42 filter paper. First 10 ml portion of the filtrate was discarded and

the resulting tablet extract (50 µg ml-1 in MPM) was subjected to analysis by

following the general procedure described under ‘calibration curve’.

Procedure for the analysis of placebo blank and synthetic mixture

A matrix substance containing starch (100 mg), acacia (100 mg), sodium

citrate (50 mg), hydroxyl cellulose (50 mg), magnesium stearate (20 mg), talc

(150 mg) and sodium alginate (10 mg) was prepared (by assuming them as

21

adjuvants added to tablets) by mixing all the components into a homogeneous

mixture. A 50 mg of the placebo blank was accurately weighed and its solution

was prepared as described under ‘procedure for tablets’, and then subjected to

analysis by following the general procedure.

A synthetic mixture was prepared by adding an accurately weighed 50 mg

of MPM to the placebo mentioned above. A portion containing 5 mg MPM was

subjected to extraction procedure described for tablets to prepare 50 µg ml-1 MPM

solution. A 3 ml aliquot of the resulting synthetic mixture solution (15 µg ml-1)

was subjected to the analysis (n=5) by following the general procedure.

2.1.3 RESULTS AND DISCUSSION

The proposed method is indirect and is based on the determination of

unreacted cerium(IV) after the reaction between MPM and the oxidant is ensured

to be complete; and relies on a well known reaction which is shown below:

MPM + H+ Oxidation product of MPM

Ce(IV)(Known excess)

+ Unreacted Ce(IV)

Unreacted Ce(IV)

p-DMAB in HClO4 medium

Orange coloured product measured at 460 nm

+

The unreacted Ce4+ was treated with p-DMAB in HClO4 medium to yield

formic acid and p-dimethylaminophenol, which upon further oxidation gave the

corresponding quinoimine derivative [43]. The possible reaction scheme resulting

in the formation of coloured chromogen is given below:

22

p-DMAB

O

N

H2O

N

OHHO

+Ce4+

-e-

N+

O

OH

H

+Ce4+

-e-

O

N

O

+H2OHCOOH

OH

N

2Ce4+

-2e-

O

N+

(measured at 460 nm)

+4Ce3+

Scheme 2.1.1 The possible reaction pathway for the oxidation of p-DMAB by

Ce(IV) in the presence of HClO4 .

2.1.3.1 METHOD DEVELOPMENT

Absorption spectra

The reaction product of p-DMAB with Ce(IV) is yellowish red coloured

quinoimine derivative peaking at 460 nm; MPM and p-DMAB had no absorption

at 460 nm. The decrease in the absorption intensity at 460 nm, caused by the

presence of the drug, was directly proportional to the concentration of the drug

reacted. Figure 2.1.1 illustrates the absorption spectra of the reaction product

formed due to the reaction between Ce(IV) and p-DMAB in the presence of

different concentrations of MPM and in the absence of MPM.

Optimization of reaction variables

Selection of reaction medium

A 4 M HClO4 medium was found necessary for rapid and quantitative

reaction between MPM and Ce(IV), and to obtain maximum and constant

absorbance. The reaction is more rapid in HClO4 medium rather than other acids

due to its maximum oxidation potential. The oxidation potential values of Ce(IV)

in HClO4, H2SO4, HNO3, and HCl are 1.75, 1.44, 1.61 and 1.28 V, respectively

[44]. Therefore, all the solutions [MPM, Ce(IV) and p-DMAB] were prepared in 4

23

M perchloric acid through out the investigation and the same was maintained as

reaction medium.

Figure 2.1.1 Absorption spectra recorded for oxidation product of p-DMAB in the

presence of different concentrations of MPM and in the absence of MPM. a.Blank, b.5 µg ml-1 MPM, c. 20 µg ml-1 MPM and d. 25 µg ml-1 MPM.

Optimization of Ce(IV) concentration

To fix the optimum concentration of Ce4+, different concentrations of

oxidant were reacted with a fixed concentration of p-DMAB in HClO4 medium

and the absorbance measured at 460 nm. A constant and maximum absorbance

resulted with 30 µg ml-1 Ce4+ and, hence, different concentrations of MPM were

reacted with 1 ml of 300 µg ml-1 Ce4+ in HClO4 medium before determining the

residual Ce4+ via the reaction scheme illustrated earlier. This facilitated the

optimization of the linear dynamic range over which procedure could be applied

for the assay of MPM.

Study of reaction time and stability of the coloured species

Under the described experimental conditions, the reaction between MPM

and Ce4+ was complete within 10 minutes at room temperature (28±2 °C). After

the addition of p-DMAB, a standing time of 15 min was necessary for the

formation of coloured product, and thereafter, the absorbance of the coloured

product (quinoimine derivative) was stable for more than an hour.

Effect of diluent

In order to select proper solvent for dilution, different solvents were tried.

The highest absorbance values were obtained when 4 M HClO4 was used as

diluting solvent. Substitution of 4 M HClO4 by other solvents (methanol, water, 6

M HClO4) resulted in decrease in the absorbance values.

24

2.1.3.2 METHOD VALIDATION

Linearity and sensitivity

The measured absorbance values for the concentration range of 1.25 – 30.0

µg ml-1 MPM produced a inverse linear curve. The graph is described by the

regression equation:

Y = a + bX

(where Y = absorbance of 1-cm layer of solution; a = intercept; b = slope and X =

concentration in µg ml-1). Regression analysis of the Beer’s law data using the

method of least squares was made to evaluate the slope (b), intercept (a) and

correlation coefficient (r) for each system and the values are presented in Table

2.1.1. The optical characteristics such as Beer’s law limit, molar absorptivity and

Sandell sensitivity [45] values are given in Table 2.1.1.The limits of detection

(LOD) and quantification (LOQ), calculated according to ICH guidelines [46]

using the formulae:

LOD = 3.3 S/b and LOQ = 10 S/b

(where S is the standard deviation of seven blank absorbance values, and b is the

slope of the calibration plot) are also presented in Table 2.1.1 and reveal high

sensitivity of the proposed method.

Table 2.1.1 Regression and quantitative parameters

Parameters Value

max, nm 460

Color stability, min > 1h

Linear range, µg ml-1 1.25 – 30.0

Molar absorptivity, L mol-1 cm-1 1.28 × 104

Sandell sensitivity*, µg cm-2 0.034

Limit of detection, µg ml-1 0.56

Limit of quantification, µg ml-1 1.7

Regression equation, Y** -0.9968

Intercept (a) 0.9553

Slope (b) -0.0287 *Limit of determination as the weight in µg ml-1 of solution, which corresponds to an absorbance of A = 0.001 measured in a cuvette of cross-sectional area 1 cm2 and l = 1 cm. **Y = a + bx, where y is the absorbance and x is concentration in µg ml-1.

25

Accuracy and precision

The repeatability of the proposed method was determined by performing

replicate determinations. The intra-day and inter-day variation in the analysis of

MPM was measured at three different levels. The accuracy of an analytical

method expresses the closeness between the reference value and the found value.

Accuracy was evaluated as percentage relative error between the measured and

taken concentrations. The results of this study are compiled in Table 2.1.2 and

speak of the fair intermediate precision (RSD ≤ 3.14%) and accuracy (RE ≤

3.64%) of the results.

Table 2.1.2 Results of intra-day and inter-day accuracy and precision study

MPM taken,

µg ml-1

Intra-day

accuracy and precision

Inter-day

accuracy and precision

MPM found,

µg ml-1

RE,

%

RSD,

%

MPM found,

µg ml-1

RE,

%

RSD,

%

10.0

15.0

20.0

10.12

15.13

20.26

1.15

0.86

1.28

1.58

1.04

1.14

10.22

15.55

20.53

2.18

3.64

2.65

2.34

2.85

3.14

RE. relative error, RSD. relative standard deviation Selectivity

In the analysis of placebo blank, the absorbance value was same as that of

the reagent blank and this confirmed the non-interference by the inactive

ingredients added to prepare the placebo.

In the analysis of synthetic mixture, a 3 ml aliquot of 50 µg ml-1 MPM was

subjected to analysis (n = 5). It was found that 97.36% MPM was recovered with

standard deviation of 1.66%. These results complement the findings of the

placebo blank analysis with respect to selectivity.

Robustness and ruggedness

To evaluate the robustness of the method, the reaction time and volume of

p-DMAB were deliberately altered incrementally. To check the ruggedness,

analysis was performed by four different analysts; and using three different

cuvettes by the same analyst. The robustness and the ruggedness were checked at

three different drug levels. The intermediate precision, expressed as percent RSD,

26

which is a measure of robustness and ruggedness was within the acceptable limits

(0.58 – 2.65%) as shown in the Table 2.1.3.

Table 2.1.3 Results of robustness and ruggedness study expressed as intermediate precision (%RSD)

Application to tablets

Commercial MPM tablets were analyzed using the developed method and

also a reference BP method [5]. The reference method involved the potentiometric

titration of MPM with 0.1 M HClO4 in anhydrous acetic acid medium. The results

obtained by the proposed method 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 [47] with those of the

reference method at 95 % confidence level as summarized in Table 2.1.4. The

results showed that the calculated t-and F-values did not exceed the tabulated

values inferring that proposed methods are as accurate and precise as the reference

method.

Table 2.1.4 Results of analysis of cellcept tablet by the proposed methods and statistical comparison of the results with the official method.

MPM studied µg ml-1

Robustness (RSD, %)

Ruggedness (RSD, %)

Conditions altered*

Inter-analysts (n=4)

Inter-cuvettes (n = 4)

Volume of

HClO4

(n=3)

Reaction

time

(n=3)

10.0

15.0

20.0

0.76

1.15

0.84

1.06

0.72

0.68

0.62

0.58

0.74

2.65

1.74

2.38

*Volume of HClO4 varied was 1±0.1 ml; reaction time varied was 15±1 min after adding p-DMAB

Tablet analyzed

Label claim, mg/tablet

Found* (Percent label claim ±SD)

Official BP method Proposed method

CellCept

500 500 98.33±1.14

99.02±2.75

t = 0.56

F = 5.82

27

Recovery study

To further ascertain the accuracy and reliability of the method, recovery

experiments were performed via standard-addition procedure. Pre-analyzed tablet

powder was spiked with pure MPM at three different levels and the total was

found by the proposed method. Each determination was repeated three times. The

percent recovery of pure MPM added (Table 2.1.5) was within the permissible

limits indicating the absence of interference from inactive ingredients in the assay

procedure.

Table 2.1.5 Results of accuracy assessment by recovery experiments

Tablets studied MPM in tablet,

µg ml-1

Pure MPM added, µg ml-1

Total found, µg ml-1

Pure MPM recovered*, Percent±SD

CellCept 500

4.95

4.95

4.95

5.0

10.0

15.0

10.25

15.49

19.58

106.0±1.12

105.4±2.12

97.53±2.21

*Mean value of three measurements

28

Section 2.2

SPECTROPHOTOMETRIC ASSAY OF MYCOPHENOLATE MOFETIL

IN PHARMACEUTICALS USING FOLIN-CIOCALTEU AND

FERRICYANIDE-FERRIC CHLORIDE REAGENTS

2.3.1 INTRODUCTION

Folin-Ciocalteu’s (FC) reagent or more commonly known as F-C reagent

is named after two chemists, Otto Folin and Vintila Ciocalteu, who first used the

reagent for the determination of tyrosine and tryptophane in proteins [48]. F-C

reagent is a mixture of acids and involves the chemical species

3H2O.P2O5.13WO3.5MoO3.10H2O and 3H2O.P2O5.14WO3.4MoO3.10H2O. Many

organic compounds containing basic nitrogen moiety and phenolic groups are

known to form water soluble blue-coloured compound, molybdenum blue when

they react with Folin-Ciocalteu’s reagent [49-52] in solutions rendered alkaline

with sodium carbonate. This is widely used for the colorimetric assay of phenolic

and polyphenolic antioxidants [52].

F-C reagent is used extensively in the determination of large number of

substances of pharmaceutical interest such as naproxen, oxyphenbutazone,

mefenamic acid, indomethacin, diclofenac sodium [53], ampicillin, amoxycillin,

and carbenicillin [54], hydralazine [55], cefotaxime and ceftriaxone [56], ajmaline

and brucine [57], aceclofenac and indapamide [58], tinofovir [59], isoxsuprine

hydrochloride [60], diacerein [61], doxycycline [62] and buspirone [63].

Likewise, amines also react with iron(III) chloride in the presence of

ferricyanide to form intensely colored Prussian blue [64] and this reaction has

been the basis for the assay of many drugs [65-68].

The analytical utility of FC and ferricyanide-ferric chloride (FFC) reagents

cited above and the literature survey presented previously reveal that these

reagents have not been used before for the spectrophotometric assay of MPM. The

author has been successful in developing two simple and sensitive

spectrophotometric methods for the determination of MPM in pharmaceuticals

using these two reagent systems. The method development procedure, validation

results and application of the methods are presented in this section (Section 2.2).

29

2.2.2 EXPERIMENTAL

2.2.2.1 Apparatus

The instrument used for absorbance measurement is the same as described

in Section 2.1.2.1.

2.2.2.2 Materials

Distilled water was used throughout the work. Folin-Ciocalteu reagent

(Merck, Mumbai, India), potassium ferricyanide (Glaxo Laboratory, Mumbai),

ferric chloride (Loba Chemie Ltd, Mumbai, India), citric acid (Surabhi Chemicals,

Baroda, India), sodium lauryl sulphate (Loba Chemie Ltd, Mumbai, India),

sodium carbonate (S.D. Fine Chem Ltd, Mumbai, India), sodium acetate (Merck,

Mumbai, India) and concentrated hydrochloric acid (sp. Gr. 1.18; Merck,

Mumbai, India) used were of analytical reagent grade or chemically pure grade

and used without further purification. Pure MPM and its tablets used were the

same described in Section 2.1.2.

2.2.2.3 Reagents and solutions

Hydrochloric acid (2 M & 0.1 M): A 2 M HCl was prepared by diluting

concentrated acid (Merck, Mumbai, India, Sp, Gr, 1.18) with water. It was further

diluted with water to get 0.1 M acid.

Folin-Ciocalteu (F-C) reagent (1:1 v/v): Prepared by mixing 125 ml of

analytical grade F-C reagent with 125 ml of water.

Sodium carbonate (Na2CO3, 20% w/v): Prepared by dissolving 20 g of pure

sodium carbonate in 100 ml of water. It was filtered before use.

Sodium lauryl sulphate (SLS, 1%): Prepared by dissolving 1 g in 100 ml of

water.

Potassium ferricyanide-citric acid mixture (0.2% each): Prepared by

dissolving the mixture containing 200 mg each of pure potassium ferricyanide and

citric acid in 100 ml of water.

Sodium acetate (NaOAc, 6%): Six g of the compound was dissolved in and

diluted to 100 ml with water.

Ferric chloride (FeCl3, 0.5%): Prepared by dissolving required amount of the

salt in water containing few drops of 2 M HCl.

Standard MPM Solution

A stock standard solution of MPM (150 µg ml-1) was prepared by

dissolving 15 mg of pure drug in 20% Na2CO3 and the solution was made up to

30

the mark with the same solvent and used for the assay in F-C method. For FFC

method, a 400 µg ml-1 MPM solution was prepared in 0.1 M HCl and 10 ml of

resulting solution was diluted to 100 ml with the same solvent in a volumetric

flask to get a working concentration of 40 µg ml-1.

2.2.2.4 General procedures

F-C method (using F-C reagent)

Different aliquots of standard MPM solution (150 µg ml-1) ranging from

0.2-2.0 ml were transferred into a series of 10 ml of volumetric flasks and the total

volume was brought to 2 ml with 20% Na2CO3. To each flask, 3 ml of 1:1 F-C

reagent, 3 ml of 20% Na2CO3 and 1 ml of 1% SLS solutions were successively

added. The flasks were stoppered, content mixed well and kept at room

temperature for 10 min. The volume was made upto the mark with water and the

absorbance of each solution was measured at 770 nm against a reagent blank

similarly prepared in the absence of MPM.

FFC method (using ferricyanide and ferric chloride)

Varying volumes of 40 µg ml-1 MPM solution (equivalent to 0.8 – 16.0 µg

ml-1 MPM) were transferred into a series of 10 ml volumetric flasks and the total

volume was brought to 4 ml by adding 0.1 M HCl. Then, 1 ml each of

ferricyanide-citric acid reagent (0.2% in each) and 0.5% FeCl3 solutions were

accurately added, content mixed and the flasks were kept at room temperature for

15 min. The volume in each flask was made up to the mark with 6% NaOAc

solution and after mixing, the absorbance was measured at 730 nm against reagent

blank.

In both methods, standard graphs were prepared by plotting the absorbance

versus MPM concentration, and the concentration of the unknown was read from

the calibration graph or computed from the respective regression equation derived

using the absorbance-concentration data.

Procedure for tablets

An amount of finely ground tablet powder equivalent to 15 mg of MPM

was accurately weighed and transferred into a 100-ml volumetric flask, the flask

was shaken with ~70 ml of 20% Na2CO3 for about 20 min; and finally volume

was made upto the mark with the same solvent. The content was kept aside for 5

min, and filtered using Whatman No. 42 filter paper. First 10 ml portion of the

31

filtrate was discarded and a suitable aliquot (say 1 or 1.5 ml) was used for assay in

F-C method as described earlier.

The tablet powder equivalent to 40 mg MPM was taken in a 100 ml

volumetric flask and about 70 ml of 0.1 M HCl was added. The flask was shaken

for ~20 min and the volume was completed up to the mark by adding 0.1 M HCl.

After filtering, the resulting extract was used for the assay in FFC method by

following the general procedure after appropriate dilution.

Procedure for the analysis of placebo blank

A placebo blank was prepared as described in the previous section. Fifty

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.

Procedure for the analysis synthetic mixture

An accurately weighed 100 mg of MPM was added to 200 mg of placebo

blank and homogenized. Synthetic mixture equivalent to 15 and 4 mg MPM was

separately weighed out into two different 100 ml volumetric flasks and the

extracts were prepared as described under the general procedure for tablets.

Suitable aliquots of the resulting 150 µg ml-1 (F-C method) and 40 µg ml-1 (FFC

method) solutions were analyzed at three levels by following the general

recommended procedures.

2.2.3 RESULTS AND DISCUSSION

The proposed methods are based on the redox reaction between the drug

and either F-C reagent or ferricyanide-ferric chloride systems. In the F-C method,

reaction follows the reduction of phospho-molybdo tungstic mixed acid of the F-C

reagent [48] by MPM, in the presence of sodium carbonate, and the resulting blue

chromogen was measured at 770 nm. The colour formation by F-C reagent with

MPM may be explained based on the analogy with reports of earlier workers [69-

72]. The mixed acids in the F-C reagent are the final chromogen and involve the

following chemical species:

3H2O•P2O5•13WO3•5MoO3•10H2O and 3H2O•P2O5•14WO3•4MoO3•10H2O

MPM probably effects reduction of 1, 2 or 3 oxygen atoms from tungstate

and/or molybdate in the F-C reagent, there by producing one or more possible

reduced species which have characteristic intense blue color.

32

The FFC method involves the reaction of MPM with ferric chloride, in the

presence of potassium ferricyanide, under mild acidic conditions (citric acid), to

produce a blue color with maximum absorption at 730 nm. The first step in the

colour development is the reduction of iron(III) of ferric chloride to iron(II) which

subsequently reacts with ferricyanide to form Prussian blue.

2.2.3.1 Method development

Spectral characteristics

The intensely blue coloured products formed in F-C method and FFC

method exhibited maximum absorption at 770 and 730 nm, respectively. The

absorption spectra of the blue coloured products and of the reagent blanks are

shown in Figure 2.2.1.

Figure 2.2.1 Absorption spectra of: a. F-C method reaction product (20.0 µg ml-1

MPM); b. F-C method blank; c. FFC method Prussian blue product

(8.0 µg ml-1 MPM) and d. FFC method blank.

Optimization of experimental variables

The optimum experimental conditions were established by variation of one

variable at a time, and observing its effect on the absorbance of the coloured

species.

F-C method

Selection of reaction medium

The reaction was tried in different aqueous bases such as borax, sodium

hydroxide, sodium carbonate, sodium bicarbonate, sodium acetate and sodium

hydrogen phosphate. The best results were obtained when Na2CO3 was used. In

order to determine the optimum concentration of base, different volumes of 20%

Na2CO3 solution (0.5 to 5 ml) were used with a fixed concentration of MPM.

From the results (Figure 2.2.2), it is clear that 3 ml of 20% sodium carbonate

33

solution was found optimum. This is in addition to Na2CO3 present in the drug

solution.

Figure 2.2.2 Effect of Na2CO3 concentration on the absorbance of coloured

species (30 µg ml-1 MPM).

Effect of F-C reagent concentration

To study the effect of F-C reagent concentration on the absorbance,

varying volumes of 1:1 F-C reagent (1 to 5 ml) were added to a fixed

concentration of MPM. The results revealed that 3 ml of reagent produced

maximum absorbance (Figure 2.2.3). Hence, 3 ml of 1:1 F-C reagent in a total

volume of 10 ml were used throughout the investigation.

Figure 2.2.3 Effect of F-C reagent concentration on the absorbance of the

coloured species (30 µg ml-1 MPM).

Importance of addition of SLS

The blue coloured chromogen formed in alkaline medium was not stable

for longer period and flocculation of the solution was observed. In order to avoid

this, different volumes of 1% SLS were introduced. The absorbance of coloured

34

product was stable and no solid particles formed in the presence of 0.75 to 2 ml of

1% SLS. Therefore, a 1 ml of 1% SLS was used in the investigation.

Reaction time and stability of the coloured species

The reaction was not instantaneous. Maximum color was developed in 10

min after mixing the reactants and was stable for at least 50 min thereafter.

Effect of order of addition of reactants

Different results were obtained when different orders of additions of

reactants were followed. The order of addition of reactants followed in the

recommended procedure resulted in rapid color formation with maximum

sensitivity and stability.

FFC method

Selection of reaction medium

Color formation was slow and blank also yielded color when different

reaction media including HCl, H2SO4, H3PO4 and acetic acid were employed. This

problem was overcome by introducing citric acid to ferricyanide reagent solution

[73].

Effect of concentration of ferricyanide-citric acid and FeCl3

To optimize the concentrations of ferricyanide-citric acid and ferric

chloride reagents, different volumes of these reagents were used with a fixed

concentration of MPM. Volumes in the range 0.75-1.5 ml each of ferricyanide-

citric acid (0.2% in each) and 0.5% FeCl3 were found necessary to achieve

maximum color formation in a reasonable time. Hence, 1 ml each of the two

reagent systems were employed in the final study.

Effect of sodium acetate on the stability of Prussian blue color

Under the optimized reaction conditions of time, the absorbance continued

to increase slowly and no constant absorbance resulted even after 60 min. In order

to stabilize the Prussian blue color, sodium acetate solution was added as

recommended by Genius [74]. When the volume was diluted to the mark with 6%

NaOAc, the reaction was completely arrested and the measured absorbance was

found to be stable for upto 30 min, thereafter.

35

2.2.3.2 Method validation

Linearity, sensitivity, limits of detection and quantification

A linear correlation was found between absorbance at max and

concentration of MPM in the ranges given in Table 2.2.1. Regression analysis of

the Beer’s law data using the method of least squares was made to evaluate the

slope (b), intercept (a) and correlation coefficient (r) for each system and the

values obtained from this investigations are presented in Table 2.2.1. The optical

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

sensitivity values [45] of both the methods are also given in Table 2.2.1. The high

values of ε and low values of Sandell sensitivity and LOD indicate the high

sensitivity of the proposed methods.

Table 2.2.1 Sensitivity and regression parameters

Parameter F-C method FFC method

max, nm 770 730

Color stability, min 50 30

Linear range, µg ml-1 3-30 0.8-16

Molar absorptivity(ε), l mol-1cm-1 1.06 × 104 2.91 × 104

Sandell sensitivity*, µg cm-2 0.0408 0.0149

Limit of detection (LOD), µg ml-1 0.79 0.04

Limit of quantification (LOQ), µg ml-1 2.39 0.13

Regression equation, Y**

Intercept (a) 0.0146 -0.0136

Slope (b) 0.0228 0.0692

Regression coefficient (r) 0.9995 0.9983 *Limit of determination as the weight in µg ml-1 of solution, which corresponds to an absorbance of A = 0.001 measured in a cuvette of cross-sectional area 1 cm2 and l = 1 cm. **Y=a+bX, Where Y is the absorbance, X is concentration in µg ml-1 , a is intercept and b is slope

Accuracy and precision

The precision and accuracy of the proposed methods were studied by

repeating the experiment seven times within the day to determine the repeatability

(intra-day precision) and five times on different days to determine the

intermediate precision (inter-day precision). The assay was performed for three

levels of analyte in each method. The results of this study are summarized in

Table 2.2.2. The percentage relative standard deviation (RSD, %) values were ≤

36

2.31% (intra-day) and ≤ 3.56% (inter-day) indicating good precision of the

methods. Accuracy was evaluated as percentage relative error (RE, %) between

the measured mean concentrations and taken concentrations of MPM, and it was ≤

2.71% (intra-day) and ≤3.56 % (inter-day) demonstrating the accuracy of the

proposed methods.

Table 2.2.2 Results of intra-day and inter-day accuracy and precision study

Selectivity

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

absorbance by analyzing the placebo blank. In the analysis of placebo blank

solution the absorbance in each case was equal to the absorbance of blank which

revealed no interference. To assess the role of the inactive ingredients on the assay

of MPM, the general procedure was applied on the synthetic mixture extract by

taking three different concentrations of MPM: 10, 20 and 30 µg ml-1 in F-C

method and 4, 8 and 12 µg ml-1 in FFC method. The percentage recovery values

were in the range 95.4 – 107.3% with RSD < 4% indicating clearly the non-

interference from the inactive ingredients in the assay of MPM.

Robustness and ruggedness

The robustness of the methods was evaluated by making small incremental

changes in the volumes of reactants (Na2CO3 in F-C method; ferricyanide-citric

acid in FFC method) and reaction times, and the effects of the changes were

studied by measuring the absorbance of the coloured products. The changes had

negligible influence on the results as revealed by small intermediate precision

Method MPM taken,

µg ml-1

Intra-day accuracy and precision

(n=7)

Inter-day accuracy and precision

(n=5) MPM found, µg ml-1

RE, %

RSD, %

MPM found, µg ml-1

RE, %

RSD, %

F-C

15.0

22.5

30.0

15.39

22.92

30.77

2.58

1.85

2.57

1.59

0.50

1.20

15.33

23.00

30.87

2.20

2.22

2.90

2.35

1.89

3.56

FFC

4.0

8.0

12.0

4.08

7.86

11.67

2.56

1.71

2.71

2.31

1.18

0.85

4.08

8.15

12.43

2.00

1.88

3.58

2.89

2.22

3.56 RE. relative error, RSD. relative standard deviation

37

values expressed as RSD (≤ 2.11%). Method ruggedness was demonstrated by

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

analysis on three different cuvettes in the same laboratory. Intermediate precision

values (RSD, %) in both instances were in the range 1.78-2.58% indicating

acceptable ruggedness. These results are presented in Table 2.2.3.

Table 2.2.3 Results of method robustness and ruggedness study expressed as intermediate precision (RSD, %)

Method MPM taken,

µg ml-1

Robustness Ruggedness Parameters altered Inter-

analysts RSD, %

(n=4)

Inter-cuvettes RSD, %

(n=4)

Volume of

reactants*

Reaction

timeΨ

F-C

15.0

22.5

30.0

1.58

1.13

1.52

0.89

1.04

1.70

2.11

1.89

1.78

1.86

2.08

2.22

FFC

4.0

8.0

12.0

1.95

2.04

1.88

2.11

2.00

1.86

2.23

2.58

1.99

1.88

2.45

2.22

*The volumes reactant were 3±0.2 ml of Na2CO3 in F-C method and 1±0.1 ml of ferricyanide-citric acid in FFC method. ΨThe reaction times were 10±1 and 15±2 min, in F-C and FFC methods, respectively. Application to tablets

The proposed methods were applied to the quantification of MPM in

commercial tablets. The tablets were assayed by the reference method [5]. The

results obtained by the proposed methods agreed well with the label claim and

also are in agreement with those by the reference method. The results obtained

were compared statistically as described in Section 2.1.3.2. The results of assay

are given in Table 2.2.4.

Table 2.2.4 Results of analysis of tablets by the proposed methods and statistical comparison of the results with the reference method

Tablet brand name

Nominal amount

(mg/tablet)

Found* (Percent of label claim ± SD) Reference

method Method A Method B

CellCept 500 500 99.04±1.32

98.64±1.85

t = 0.39

F = 1.96

97.86±2.08

t = 1.09

F = 2.48 *Mean value of five determinations.

38

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 MPM with pure MPM at three different levels (50,

100 and 150 % of the content present in the preparation and the total was found by the proposed methods. Each test was repeated

three times. In both the cases, the recovery percentage values ranged between 96.4 and 101.5% with standard deviation in the range

1.58-2.63%. Closeness of the results to 100% showed the fairly good accuracy of the methods. The results are shown in Table 2.2.5.

Table 2.2.5 Results of recovery study via standard-addition method

F-C method FFC method

MPM in tablet, µg ml-1

Pure MPM added, µg ml-1

Total found, µg ml-1

Pure MPM recovered

(Percent±SD*)

MPM in tablet, µg ml-1

Pure MPM added, µg ml-1

Total found, µg ml-1

Pure MPM recovered

(Percent±SD*)

9.86

9.86

9.86

5.00

10.00

15.00

14.68

19.73

25.09

96.40±2.14

98.70±1.70

101.5±2.36

3.91

3.91

3.91

2.00

4.00

6.00

5.88

7.70

9.95

98.50±1.58

94.75±2.63

100.7±2.14 *Mean value of three determinations

39

Section 2.3

SPECTROPHOTOMETRIC DETERMINATION OF MYCOPHENOLATE

MOFETIL AS ITS CHARGE-TRANSFER COMPLEXES WITH TWO -

ACCEPTORS

2.3.1 INTRODUCTION

A charge-transfer complex (CT complex) also referred as electron-donor-

acceptor complex is one which is formed by a molecular interaction between

electron donors and acceptors, in which the attraction between the molecules is

created by an electronic transition into an excited electronic state, such that a

fraction of electronic charge is transferred between the molecular entities. The

source molecule from which the charge is transferred is called the electron donor

(D) and the receiving molecule is called the electron acceptor (A).

D + A → DA

The nature of the attraction in a charge-transfer complex is not a stable

chemical bond and is much weaker than covalent forces; rather it is better

characterized as a weak electron resonance. As a result, the excitation energy of

this resonance occurs very frequently in the visible region of the electro-magnetic

spectrum [75]. The association does not constitute a strong covalent bond and is

subject to significant temperature, concentration, and host (e.g., solvent)

dependencies.

Many pharmaceutical compounds containing amino groups have shown

appreciable tendency towards formation of these type of complexes and include

perindopril [76], barbiturates [77], clozapine [78], disopyramide [79], cefadroxil

[80], ceterizine [81], ketamine hydrochloride [82], diethylcarbamazine citrate

[83], phenobarbital sodium, thiopental sodium and fimonaric [84], tamoxifin and

methotrexate [85], pyrimethamine [86], astemizole [87], pheniramine maleate

[88], cyproheptadine, methdilazine, promethazine [89], atenolol [90], albendazole

[91] etc., to name a few.

Not a single spectrophotometric method has been reported for the assay of

MPM in pharmaceuticals. This prompted the author to exploit the amino group of

this compound to develop of two simple and rapid methods. Both methods are

based on the formation of charge-transfer complex of MPM with p-chloranilic

acid (p-CAA) or 2,3-dichloro-5,6-dicyano-p-benzoquinone (DDQ) in dioxane-

40

acetonitrile medium resulting coloured product measurable at 520 nm (p-CAA) or

580 nm (DDQ).

Acetonitrile-1,4-dioxane solvent system was the medium of choice for

many C-T complexation reactions [92-96], and the same was used by the author

for methods employing p-CAA and DDQ in the present study. The details of

method development, validation and applications are presented in this section

(Section 2.3).

2.3.2 EXPERIMENTAL

2.3.2.1 Apparatus

The instrument used for absorbance measurements was the same as

described in Section 2.1.2.1.

2.3.2.2 Materials

Spectroscopic grade 1,4-Dioxane and acetonitrile were from Merck,

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

pure MPM and its tablets used were the same described in Section 2.1.

2.3.2.3 Reagents

p-Chloranilic acid (p-CAA, 0.5%) & 2,3-Dichloro-5,6-dicyanoquinone (DDQ,

0.25%): Prepared by dissolving required amount of the pure compounds (both

S.D. Fine Chem Ltd, Mumbai) in 1,4-dioxane.

Standard MPM stock solution

For p-CAA method, a 500 µg ml-1 MPM stock solution was prepared by

dissolving 50 mg of pure drug in acetonitrile and diluting to volume in a 100 ml

volumetric flask with the same solvent, and the same was diluted with acetonitrile

to get 150 µg ml-1 and used for the assay in DDQ method.

2.3.2.4 General procedures

p-CAA method

Varying aliquots of standard MPM solution equivalent to 40 - 400 µg ml-1

(0.4 – 4.0 ml of 500 µg ml-1) were accurately transferred into a series of 5 ml

calibrated flasks and the total volume in each flask was brought to 4 ml by adding

acetonitrile. After the addition of 1 ml of 0.5% p-CAA solution, the content was

mixed well and the absorbance was measured at 520 nm after 5 min against a

reagent blank similarly prepared without adding MPM solution.

41

DDQ method

Into a series of 5 ml calibrated flasks, aliquots (0.2 – 4.0 ml) of standard

MPM solution (150 µg ml-1) equivalent to 6 - 120 µg ml-1 MPM were accurately

transferred, and to each flask 1 ml of 0.25 % DDQ solution was added and mixed.

After 5 minutes, the absorbance of the purple coloured C-T complex was

measured at 580 nm against the reference blank similarly prepared.

Standard graph was prepared by plotting the absorbance versus MPM

concentration, and the concentration of the unknown was read from the calibration

graph or computed from the respective regression equation derived using the

absorbance-concentration data.

Procedure for tablets

An amount of tablet powder equivalent to 50 mg of MPM was transferred

into a 100 ml volumetric flask and about 70 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 p-

CAA method. This tablet extract (500 µg ml-1) was diluted to 150 µg ml-1 with

acetonitrile and suitable aliquot was used for the assay by DDQ method.

Procedure for the analysis of placebo blank and synthetic mixture

Placebo blank and the synthetic mixture were prepared as described in

Section 2.1.2.4. Fifty 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 50 mg MPM was

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

equivalent to 500 µg ml-1 MPM was prepared as described under the general

procedure for tablets and used in p-CAA method. Calculated volume of the above

extract was diluted to 150 µg ml-1 with acetonitrile and used for DDQ method.

2.3.3 RESULTS AND DISCUSSION

Spectral characteristics and reaction pathway

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

acceptors, namely, p-CAA and DDQ, to produce coloured charge transfer

complexes in 1,4-dioxane-acetonitrile solvent system according to the following

equation:

42

MPM + A MPM-A MPM+

+ A.-

C-T complex Radical anion In the p-CAA method, MPM reacts with the reagent and gives a red

chromogen that exhibits a strong absorption maximum at 520 nm in dioxane-

acetonitrile medium (Figure 2.3.1). This can be attributed to the formation of

charge-transfer complex between MPM and p-CAA followed by the formation of

radical ions which probably was due to the dissociation of the original (MPM-p-

CAA) complex promoted by the high ionizing power of the acetonitrile solvent

[93].

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

acetonitrile at room temperature gave a purple colored chromogen with strong

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

anion [96] and the wavelength 580 was selected for further studies because of

higher sample absorbance and lower blank absorbance readings (Figure 2.3.1).

Figure 2.3.1 Absorption spectra of : (a) MPM-p-CAA C-T complex( b)blank,

and (c) MPM-DDQ C-T complex (d)blank

2.3.3.1 Method development

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

complexes at 520 and 580 nm, for p-CAA and DDQ method, respectively, by

varying one and fixing other parameters.

Effect of reagent concentration

To establish optimum concentrations of the reagents for the sensitive and

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

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

43

- 3 ml of 0.25% 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.

Effect of solvent to dissolve drug and reagents

To dissolve MPM, acetonitrile was preferred to chloroform,

dichloromethane, acetone, 2-propanol, 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 dissolve p-CAA and DDQ. Therefore, acetonitrile and

1,4-dioxane were chosen as solvents to dissolve MPM and the reagents,

respectively.

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

In both the methods the formation of C-T complex was complete within 5

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

stable for 5 h and 20 min, respectively.

Investigation of composition of C-T complexes

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

evaluated by following the Job’s continuous variations method [97]. The

experiments were performed by preparing and mixing equimolar solutions of drug

and reagent (p-CAA method: 4.61 × 10-4 M; DDQ method: 2.31 × 10-4 M) 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 2.3.2a 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 MPM

structure. Based on this, the reaction pathway for the formation of C-T complex is

proposed and shown in Scheme 2.3.1.

44

O

O

Cl

OH

OH

Cl O

O

Cl

OH

OH

Cl

O

O

N

N

Cl

Cl

O

O

N

N

Cl

Cl

O

O

OH OOO

N

OO

O

OH OOO

N

O

O

O

OH OOO

N

O

O

O

OH OOO

N

O

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

MPM+

.+ +

p-CAA radical anionmeasured at 520 nm

+

DDQ

MPM+.+ DDQ

DDQ radical anionmeasured at 580 nm

.-

MPM-DDQ C-T complex (1:1)

MPM

MPM

p-CAA. -

Scheme 2.3.1. Proposed reaction pathway for the formation of C-T complex

between MPM and p-CAA/DDQ.

(a) (b)

Figure 2.3.2 Job’s plots obtained for: (a) MPM-p-CAA C-T complex an

(b) MPM-DDQ C-T complex.

2.3.3.2 Method validation

Linearity, sensitivity, limits of detection and quantification

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 were evaluated as described in Section 2.1.3.3, and

they are presented in Table 2.3.1. The moderate values of ε and Sandell

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

The regression parameters are also compiled in Table 2.3.1.

45

Table 2.3.1 Sensitivity and regression parameters Parameter p-CAA method DDQ method max, nm 520 580 Color stability 5 h 20 min Linear range, µg ml-1 40-400 6-120

Molar absorptivity (ε), L mol-1cm-1 1.06 × 103 3.87 × 103

Sandell sensitivity*, µg cm-2 0.4106 0.1119 Limit of detection (LOD), µg ml-1 3.96 0.79 Limit of quantification (LOQ), µg ml-1 11.99 2.40 Regression equation, Y**

Intercept (a) 0.0100 0.0376 Slope (b) 0.0024 0.0080 Standard deviation of a (Sa) 0.0145 0.0350 Standard deviation of b (Sb) 5.3 × 10-5 4.75 × 10-4 Regression coefficient (r) 0.9995 0.9947 *Limit of determination as the weight in µg ml-1 of solution, which corresponds to an absorbance of A = 0.001 measured in a cuvette of cross-sectional area 1 cm2 and l = 1 cm. **Y=a+bX, Where Y is the absorbance, X is concentration in µg ml-1, a is intercept, b is slope. Precision and accuracy

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

methods were evaluated as described in Section 2.1.3.2. The results of this study

are summarized in Table 2.3.2. The percentage relative standard deviation (RSD,

%) values were ≤ 0.99% (intra-day) and ≤ 1.78% (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

MPM. The percentage relative error was calculated at each concentration and

these results are also presented in Table 2.3.2. Percent relative error (RE, %)

values of ≤ 1.39% demonstrates the high accuracy of the proposed methods.

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

46

precision values (RSD, %) in both instances were in the range 0.54-3.15%

indicating acceptable ruggedness. The results are presented in Table 2.3.3.

Table 2.3.3 Results of robustness and ruggedness study expressed as intermediate

precision (RSD, %)

Method MPM taken,

µg ml-1

Robustness Ruggedness Parameters altered

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

Inter-instruments (RSD, %),

(n=4)

Volume of p-CAA/DDQ*

Reaction timeΨ

A

100.0

200.0

300.0

0.94

1.36

1.27

0.58

0.65

0.42

1.28

0.84

0.85

2.42

3.15

2.76

B

45.0

75.0

105.0

0.66

0.74

1.03

0.36

0.85

0.64

0.96

0.78

0.54

1.98

2.38

1.62 *The volumes of p-CAA or DDQ added were 1±0.2. ΨThe reaction times were 5±1 min.

Table 2.3.2 Results of intra-day and inter-day accuracy and precision study

Method MPM taken,

µg ml-1

Intra-day accuracy and precision

(n=7)

Inter-day accuracy and precision

(n=5) MPM found, µg ml-1

RE,% RSD, % MPM found, µg ml-1

RE, % RSD, %

p-CAA 100.0 200.0 300.0

100.63 200.22 299.08

0.63 0.11 0.32

0.66 0.36 0.99

100.31 201.28 302.24

0.26 0.64 0.78

0.74 0.78 0.67

DDQ 45.0 75.0

105.0

44.73 76.04

105.92

0.58 1.39 0.88

0.50 0.87 0.92

44.69 76.05

103.89

0.67 1.25 1.04

0.74 1.36 1.78

RE. Percent relative error, RSD. relative standard deviation. n = Number of measurements

47

Application to tablets

The proposed methods were applied to the quantification of MPM in

commercially available CellCept 500 tablets. The results obtained were compared

with those obtained using a reference method [5]. Statistical analysis was done by

applying the Student’s t-test and F-test did not detect any significant difference in

the performance of the proposed methods compared with the reference method

with respect to accuracy and precision. The results of this study are given in Table

2.3.4.

*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-analyzed tablet powder with pure MPM 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. The

percentage recovery values were in the range of 97.37-104.1 with standard

deviation values from 0.74 to 1.08%. Closeness of results to 100% showed fairly

good accuracy of the method. These results are shown in Table 2.3.5.

Table 2.3.4 Results of analysis of CellCept 500 tablets by the proposed methods and statistical comparison of the results with the reference method

Nominal amount

(mg/tablet)

Found* (Percent of label claim ± SD) Reference

method p-CAA method DDQ method

500 100.6±0.76 101.0±1.16

t = 0.66 F = 2.33

99.89±1.74 t = 0.89 F = 5.24

48

Table 2.3.5 Results of recovery study via standard-addition method

p-CAA method DDQ method MPM

in tablet, µg ml-1

Pure MPM added, µg ml-1

Total found, µg ml-1

Pure MPM recovered

(Percent±SD*)

MPM in tablet, µg ml-1

Pure MPM added, µg ml-1

Total found,

µg ml-1

Pure MPM recovered

(Percent±SD*)

101.0

101.0

101.0

50.0

100.0

150.0

152.8

202.5

249.1

103.6±0.74

101.5±0.86

98.74±0.92

40.0

40.0

40.0

20.0

40.0

60.0

59.47

80.60

102.46

97.37±0.76

101.5±1.08

104.1±0.84 *Mean value of three determinations

49

Section 2.4

TITRIMETRIC ASSAY OF MYCOPHENOLATE MOFETIL IN NON-

AQUEOUS MEDIUM

2.4.1 INTRODUCTION

The weakly basic or acidic substances when dissolved in non

aqueous solvents, their basic or acidic property will be enhanced and thereby

makes it possible to titrate them with acid or base. It is the most common

titrimetric procedure used in many pharmacopoeial assays and serves a double

purpose: it is suitable for the titration of very weak acids and very weak bases, and

it provides a solvent in which organic compounds are soluble [98].

The most commonly used procedure for the assay of compounds

containing amino groups is the titration with perchloric acid in anhydrous/glacial

acetic acid medium.

When a weak basic substance is dissolved in acetic acid, the acetic acid

exerts its levelling effect and enhances the basic properties of the substance. It is

possible, therefore, to titrate a solution of a weak base in acetic acid with

perchloric acid in acetic acid, and obtain a sharp endpoint when attempts to carry

out the titration in aqueous solution are unsuccessful. The reaction involved in the

titration is as follows:

HClO4 + CH3COOH ⇌ CH3COOH2+ + ClO4

-

Basic-N + CH3COOH ⇌ Basic-NH+ + CH3COO-

CH3COOH2+ + CH3COO- ⇌ 2CH3COOH

Adding HClO4 + Basic-N ⇌ Basic-NH+ + ClO4-

The end point of most titrations is detected by the use of visual indicator

but the method can be inaccurate in very dilute or colored solutions. However

under the same conditions, a potentiometric method for the detection of the

equivalence point can yield accurate results without difficulty. The electrical

apparatus required consists of a potentiometer or pH meter with a suitable

indicator and reference electrode.

In the literature survey presented in Section 2.0.2 MPM has been assayed

potentiometrically using 0.1 M perchloric acid in anhydrous acetic acid medium,

and this procedure is applicable for the assay of MPM in macro level. In this

section two simple, rapid, reliable and cost-effective semi-micro titrimetric

50

methods in non-aqueous medium are described for the determination of MPM in

pharmaceuticals. In these methods, the drug dissolved in glacial acetic acid is

titrated with acetous perchloric acid (HClO4) with visual and potentiometric end

point detection, crystal violet being used as indicator for visual titration. The

details relating to the development and validation of the two methods for the assay

of MPM are presented in this section (Section 2.4).

2.4.2 EXPERIMENTAL

2.4.2.1 Apparatus

Potentiometric titration was performed with an Elico 120 digital pH meter

provided with a combined glass-SCE electrode system. The KCl of the salt bridge

was replaced with the saturated solution of KCl in glacial acetic acid [99].

2.4.2.2 Materials

All chemicals used were of analytical reagent grade. All solutions were

made in glacial acetic acid unless mentioned otherwise. Pure MPM and tablets

used were the same as described in Section 2.1.

2.4.2.3 Reagents and solutions

Perchloric acid: The stock solution of (~0.1 M) perchloric acid (S. D. Fine

Chem., Mumbai, India) was diluted appropriately with glacial acetic acid to get a

working solution of 5 mM and standardized with pure potassium hydrogen

phthalate using crystal violet as indicator [100].

Crystal violet indicator (0.1 %): Prepared by dissolving 50 mg of the dye (S. D.

Fine Chem., Mumbai, India) in 50 ml glacial acetic acid.

Standard drug solution

A stock standard solution containing 2 mg ml-1 MPM was prepared by

dissolving 200 mg of pure drug in glacial acetic acid in a 100 ml calibrated flask.

2.4.2.4 General procedures

Visual titration

An aliquot of the drug solution containing 4-20 mg of MPM was measured

accurately and transferred into a clean and dry 100 ml titration flask and the total

volume was brought to 20 ml with glacial acetic acid. Two drops of crystal violet

indicator were added and titrated with standard 5 mM perchloric acid to a blue

colour end point. An indicator blank titration was performed and corrections to the

sample titration were applied. The amount of the drug in the measured aliquot was

calculated from

51

Amount (mg) = VMwR/n

where V = volume of perchloric acid consumed (ml); Mw = relative molecular

mass of the drug; R = molarity of the perchloric acid and n = number of moles of

perchloric acid reacting with each mole of MPM.

Potentiometric titration

An aliquot of the standard drug solution equivalent to 4-20 mg of MPM

was measured accurately and transferred into a clean and dry 100 ml beaker and

the solution was diluted to 25 ml by adding glacial acetic acid. The combined

glass-SCE (modified) system was dipped in the solution. The content was stirred

magnetically and the titrant (5 mM HClO4) was added from a microburette. Near

the equivalence point, titrant was added in 0.1 ml increments. After each addition

of titrant, the solution was stirred magnetically for 30 s and the steady potential

(e.m.f) was noted. The addition of titrant was continued until there was no

significant change in potential on further addition of titrant observed. The

equivalence point was determined by plotting the titration curves (volume of

titrant versus e.m.f; first derivative curve or second derivative curve). The amount

of the drug in the measured aliquot was calculated as described under visual

titration.

Procedure for tablets

An amount of powder equivalent to 200 mg of MPM was weighed

accurately into 100 ml calibrated flask, 30 ml of acetone was added, shaken for

about 10 min and the extract was filtered. The procedure was repeated with 30 ml

more of acetone and the combined filtrate was kept on a water bath to evaporate

acetone. The residue was dissolved in and made up to 100 ml with glacial acetic

acid in a volumetric flask. A suitable aliquot was assayed by following the general

procedures described for visual and potentiometric end point detection.

2.4.3 RESULTS AND DISCUSSION

In the present titrimetric methods, the weakly basic property of MPM was

enhanced due to the non-levelling effect of glacial acetic acid and titrated with

perchloric acid with visual and potentiometric end point detection. Crystal violet

gave satisfactory end point for the concentrations of analyte and titrant employed.

A steep rise in the potential was observed at the equivalence point with

potentiometric end point detection (Figure 2.4.1). With both methods of

equivalence point detection, a reaction stoichiometry of 1:1 (drug:titrant) was

52

obtained which served as the basis for calculation. Using 5 mM perchloric acid, 4-

20 mg of MPM was conveniently determined.

(a) (b)

(c)

Figure 2.4.1 Plots for the titration of 12 mg MPM with 5 mM HClO4 (a) Normal titration curve, (b) First-derivative curve and (c) Second-derivative curve

2.4.3.1 METHOD VALIDATION

Accuracy and precision

Three different amounts of MPM within the range of study in each method

were analyzed in seven and five replicates in visual and potentiometric methods,

respectively, during the same day (intra-day precision) and five consecutive days

(inter-day precision). For inter-day precision, each day analysis was performed in

triplicate and pooled-standard deviation was calculated. The RSD values of intra-

day and inter-day studies for MPM showed that the precision of the methods was

good (Table 2.4.1). The accuracy of the methods was determined by the percent

mean deviation from known amount, and results are presented in Table 2.4.1.

53

Table 2.4.1 Results of intra-day and inter-day accuracy and precision study

Method

MPM taken,

mg

Intra-day accuracy and

precision, (n=7)

Inter-day accuracy and

precision, (n=5) MPM found,

mg RE,% RSD,%

MPM found,

mg RE,% RSD,%

Visual titration

6.0 12.0 18.0

5.94 11.89 17.76

1.00 0.92 1.33

1.64 0.87 1.13

6.07 12.10 18.21

1.16 0.83 1.17

0.96 1.14 1.36

Potentiometric titration

6.0 12.0 18.0

6.05 12.03 17.94

0.83 0.30 0.33

1.56 0.78 1.31

6.06 12.07 18.08

1.00 0.58 0.43

1.12 1.42 1.36

RE.relative error, RSD. relative standard deviation Ruggedness of the methods

Method ruggedness was expressed as the RSD of the same procedure

applied by four different analysts as well as using four different burettes. The

inter-analysts RSD were ≤1.04% whereas the inter-burettes RSD for the same

MPM amounts ranged from 0.42 – 1.26% suggesting that the developed methods

were rugged. The results are shown in Table 2.4.2.

Application to tablets

When the drug in the tablet was extracted with glacial acetic acid, assay

results indicated positive interference from some of the inactive ingredients. This,

however, was over come by replacing acetic acid with acetone as the extractant,

and reconstituting in acetic acid after evaporating acetone. The same tablets were

analyzed by an established procedure [6] for comparison. The reference method

consisted of the measurement of the absorbance of the tablet extract in 0.1 M HCl

at 250 nm. The recovery values of MPM obtained from this method were in the

range of 97.33 – 100.2% with standard deviation of <2%. The results obtained by

the proposed methods agreed well with those of reference method [6] and with the

label claim. The results were also compared statistically as described in Section

2.1.3.2. The results are summarized in Table 2.4.3. The results showed that the

calculated t-and F-values did not exceed the tabulated values inferring that

proposed methods are as accurate and precise as the reference method.

54

Table 2.4.2 Results of ruggedness expressed as intermediate precision (RSD, %)

Method MPM taken, mg

Ruggedness Inter-analysts

(RSD, %): (n=4)

Inter-burettes (RSD, %):

(n=4)

Visual

titration

6.0

12.0

18.0

1.04

0.84

0.72

1.26

1.04

0.94

Potentiometric

titration

6.0

12.0

18.0

0.66

0.42

0.72

1.02

0.78

0.42

Table 2.4.3 Results of analysis of tablets containing MPM by the proposed methods and comparison with the established method

*Average of five determinations

Recovery study

Accuracy and the reliability of the methods were further ascertained by

performing recovery experiments. To a fixed amount of drug in formulation (pre-

analysed): pure drug at three different levels was added, and the total was found

by the proposed methods. Each test was repeated three times. The results

compiled in Table 2.4.4 show that recoveries were in the range from 95.86 –

103.5% indicating that commonly added excipients to tablets such as talc, starch,

gelatin, sodium alginate, magnesium stearate, calcium gluconate and calcium

dihydrogen orthophosphate, did not interfere in the determination, after extraction

with acetone.

Brand name

Label claim,

mg/tablet

Found* (Percent of label claim ± SD)

Established method

Proposed methods Visual

titration Potentiometric

titration

CellCept 500

500 98.86±1.26 100.2±0.80

t = 2.06 F = 2.48

97.33±0.85 t = 2.29 F = 2.19

55

Table 2.4.4 Results of recovery study using standard addition method

Visual end point detection

Potentiometric end point detection

Tablet studied

MPM in tablet extract,

mg

Pure MPM added,

mg

Total MPM found,

mg

Pure MPM recovered*%

MPM in tablet extract,

mg

Pure MPM added,

mg

Total MPM found,

mg

Pure MPM recovered*%

CellCept 500

6.01 6.01 6.01

3.0 6.0 9.0

9.08 12.09 15.33

102.3±1.28 101.4±0.96 103.5±1.02

5.84 5.84 5.84

3.0 6.0 9.0

8.77 11.92 14.47

97.58±0.84 101.3±0.72 95.86±0.64

*Mean value of three determinations

56

Section 2.5

SUMMARY AND CONCLUSIONS-Assessment of methods

Two titrimetric and five spectrophotometric methods were developed and

validated for the assay of MPM in pharmaceuticals. The performance

characteristics of the methods developed and those of the existing methods are

compiled in Table 2.5.1 below. The BP method [5] which is based on the

potentiometric titration of drug with 0.1 M HClO4 is applicable for macroscale

assays and unsuitable for semimicro analysis. To fill this void, two methods using

acetous 5 mM HClO4 as titrant were developed. Here, the end points was detected

either visually or potentiometrically. The methods are applicable over 4-20 mg of

MPM. The methods were applied successfully to the determination of MPM in

tablets. Compared to all reported methods for MPM, the proposed methods have

two additional advantages of simplicity of operations and low-cost per analysis.

These advantageous features advocate their use in quality control laboratories for

routine use. It should be pointed out that the non-aqueous titrimetric procedures

cannot be applied directly to tablet preparations since interference from some

excipients was encountered. However, this could be overcome by extracting the

drug with acetone and reconstituting the sample with acetic acid after evaporating

acetone.

Table 2.5.1 Comparison of performance characteristics of proposed methods with

the existing methods.

Titrimetry

Sl. NO

Reagent/s Methodology Range Remarks Ref

1 HClO4 Drug titrated with 0.1 M HClO4 in anhydrous CH3COOH potentiometrically

-

Unsuitable for micro and semi micro scale

5

2 HClO4 Drug titrated with 5mM HClO4 in anhydrous CH3COOH both visually and potentiometrically

4-20 mg

Suitable for semi micro analysis

Present work

57

Spectrophotometry

Sl. NO

Reagent/s Methodology Linear range (µg ml-1)

( in l mol-1 cm-1)

LOD Remarks Ref

1. a) 0.1 M HCl b)Acetate buffer of pH 4.9

Absorbance in 0.1 M HCl or buffer of pH 4.9 measured at 250 nm.

5-40

NA

NA Less sensitive, narrow linear range

6

2 Ce(IV)-p-DMAB Absorbance of the oxidation product of p-DMAB measured at 410 nm.

1.25-30 (1.28×104)

0.56 Wide linear dynamic range, more sensitive

Present work

3 a) F-C reagent b) FFC system

Absorbance of molybdenum blue measured at 770 nm. Absorbance of Prussian blue measured at 730 nm.

3-30 (1.06×104)

0.8-16 (2.91×104)

0.79

0.04

-do- -do-

Present work

4 a) p-CAA b) DDQ

Absorbance of the radical anion formed by the dissociation of C-T complex measured at 520 and 580 nm.

40-400 (1.06×103)

6-120

(3.87×103)

3.96

0.79

Wide linear dynamic ranges, moderately sensitive, highly accurate and precise

Present work

58

Table 2.5.1 reveals that the developed spectrophotometric methods are superior to the UV-spectrophotometric methods in

terms of linear range, and sensitivity. The method using ferricyanide-ferric chloride with an value of 2.91×104 and LOD of 0.04 µg

ml-1is the most sensitive of the five methods developed. All the five methods are characterized by wide linear dynamic ranges, and

the methods using p-CAA and DDQ as reagents, based on C-T complexation reactions, though moderately sensitive ( value, 103) are

the simplest of all the methods in terms of experimental variables involved. They involve simple mixing of drug and reagent in

dioxane-acetonitrile solvent system and are free from any experimental variables that would affect their accuracy and precision; and

this is rightly reflected in their high accuracy and precision.

The method using Ce(IV)-P-DMAB involves a two-step reaction whereas the remaining methods have the distinctive feature

of a single-step reaction thereby enhancing their acceptability for routine work. The last four methods (Sl. No. 3 and 4, Table 2.5.1)

are prone to interference from nitrogenous compounds but such compounds were seldom present in the tablets. The last two methods

require the use of organic solvents though the quantity has been reduced to the barest minimum.

59

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