chapter ii titrimetric and spectrophotometric assay...
TRANSCRIPT
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.
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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.
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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
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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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