chapter 3 titrimetric, spectrophotometric...
TRANSCRIPT
CHAPTER 3
TITRIMETRIC, SPECTROPHOTOMETRIC
AND CHROMATOGRAPHIC ASSAY OF
DOXYCYCLINE HYCLATE
73
SECTION 3.0
DRUG PROFILE AND LITERATURE SURVEY
3.0.1. DRUG PROFILE
Doxycycline hyclate (DOX) is chemically known as (4S,4aR,5S,5aR,6R,12aS)-
4-(dimethylamino)-3,5,10,12,12a-pentahydroxy-6-methyl-1,11-dioxo-
1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide monohydrochloride, with ethyl
alcohol (2:1), monohydrate. Its empirical formula is (C22H24N2O8·HCl)2·C2H6O·H2O,
with a molecular weight of 1025.89. The structural formula is:
H Cl
O
OH
CONH2
NOH
O OHOH
H H
OH ½ C2H6O, ½ H2O
DOX is a yellow to light-yellow crystalline powder and also hygroscopic. It is
soluble in water and in methanol, sparingly soluble in ethanol (96 per cent). It dissolves
in solutions of alkali hydroxides and carbonates [1].
DOX is a broad spectrum antibiotic, with activity against a wide range of gram-
positive and gram-negative bacteria. It has been used for the treatment of infectious
diseases caused by rickettsiae, mycoplasmas and chlamydiae [2]. DOX is widely used
in medicine and veterinary practice. As a result, DOX residue can occur in food
products of animal origin [2].
The drug is official in the BP [1] and USP [3] which describe HPLC methods
for the determination of DOX either in raw material or in pharmaceutical formulations.
74
3.0.2.0 LITERATURE SURVEY - ANALYTICAL FRAMEWORK
3.0.2.1 Titrimetric methods
No titrimetric procedure has ever been reported for the determination of DOX
in pharmaceuticals although the technique is very simple and easily adoptable to
determine the drug content in milligram level in the quality control laboratories across
the developing countries where modern and expensive instruments are not available.
3.0.2.2 Spectrophotometric methods
Few visible spectrophotometric methods based on different reaction schemes
are found in the literature for the assay of DOX. Lopez and Calatayud developed three
methods using FIA-spectrophotometry [4]. The first method involves the measurement
of absorbance at 395 nm formed between drug and copper carbonate. The second
method was based on the oxidation of DOX using chloramine-T in alkaline medium.
The absorbance of the red colored oxidized drug was measured at 595 nm, and third
method utilizes 4-aminophenazone/potassium hexacyanoferrate(III) followed by the
measurement of dye color at 520 nm. Another method was found in the literature based
on formation of metal-ion complex by the reaction of the drug with thorium(IV) at pH
5 followed by the measurement of absorbance of yellow colored chromogen at 398 nm
[5]. The method was applicable over a concentration range of 0.4-3.2 µg ml-1
. DOX in
acetic acid medium forms a yellow colored product with sodium cobaltinitrite [6].
Absorbance was measured at 243 nm and quantification could be achieved over a
concentration range of 0.01-0.03 mg ml-1
. Saha et al, [7] developed a 1:1 complex
formation reaction with uranyl acetate in dimethyl formamide medium. Measurement
of the yellow colored chromogen was measured at 405 nm. Sunaric et al, [8] developed
a method based on the measremnt of the degraded product of DOX at 510 nm. The
reaction of the drug with H2O2 in Tris-HCl buffer of pH 8.6 at 20± 0.10C was catalysed
by Cu(II). Multivariate calibration method [9] has also been reported in DMF/NaOAc-
AcOH buffer (pH 4.5). Beer’s law was obeyed over a concentration range of 1.7-42
µg ml-1
.
75
3.0.2.3 Chromatographic techniques
Several chromatographic methods have been developed for the determination of
DOX and they include thin-layer chromatography (TLC) [10], TLC-fluorescence
scanning densitometry (TLC-FSD) [11], HPLC for body fluids [12- 18]. HPLC has
also been applied for the determination of DOX in turkey’s liver and muscle [19],
bovine tissue [14], bovine milk and muscle [20], animal tissues [21], human tissue
[22], alveolar macrophages [23] and in milk [24]. Various other chromatographic
methods have also been reported for the determination of DOX in milk and milk
powder [25], human plasma [26], human urine [27], human serum, urine, semen, tears
and saliva [28] and foods [25, 29]. DOX in pharmaceuticals has been assayed by
capillary electrophoresis [30], micellar electrokinetic chromatography [27] and HPLC
[31-36].
There are only six reports on the HPLC determination of DOX in
pharmaceuticals. The method of Snezana et al., [31] has been applied for veterinary
pharmaceutical samples by using Lichrosorb RP-8 (250 mm’4.6 mm, 10 mm particle size),
methanol:acetonitrile: 0.01M oxalic acid (2:3:5, v/v) mobile phase and at a flow rate of 1.25 ml
min-1 and detection made at 350 nm. Another HPLC method consisting of Hamilton
RP-1 (25 x 0.46, cm, i.d.); column , tetrahydrofuran:0.2M phosphate buffer (pH
8.0):0.2M tetrabutylammonium hydrogen sulphate (pH 8.0): 0.1M sodium acetate (pH
8.0): water (6:10:5:1:78) as mobile phase at a flow rate of 1 ml min-1
followed by UV-
detection at 254 nm [32].Simultaneous determination of five tetracyclines including
DOX and the impurity, 6-epi-doxycycline has been achieved using porous graphite
carbon column [33]. Two methods [34,35] have also been presented for the separation
of DOX from its analogs and for its determination in powder and tablets. HPLC
analysis of DOX in bulk drug and in dosage forms using polymeric column has been
studied by Bryan and Stewart [36].
3.0.2.4 Other techniques
Various other techniques have been reported for the in vitro and in vivo
determination of DOX and include microbiology [37], fluorimetry [38], lanthanide
sensitized luminescence spectroscopy [39], chemiluminescence spectroscopy [40],
optical fiber sensor [41], solid surface phosphorimetry [42], ion selective electrode
76
(ISE)-potentiometry [43], cyclodextrin based fluorosensor [44] and internal solid
contact sensor based on a conducting polypyrrole [45].
The literature survey presented in the foregoing paragraphs reveals no
titrimetric method for the assay of DOX. The reported spectrophotometric methods [4-
9] are not satisfactory for the routine quality assurance for one or other reason. Some of
these methods suffer from disadvantages such as poor sensitivity [4,6,7], use of organic
solvent [7,9] and scrupulous control of experimental variables and special equipment
[4-6,8]. With a view to overcome the shortcomings of the reported methods, the author
has developed titrimetric, UV and visible spectrophotometric methods employing
simple and cost-effective reagents.
The chromatographic procedures although, specific, most of the described methods
are time consuming and require multistage extraction procedures. The stability of a
drug substance or drug product is defined as its capacity to remain within established
specifications, i.e., to maintain its identity, strength, quality and purity until the retest
or expiry date [46]. There is no reported stability-indicating analytical method for the
determination of DOX in the presence of its degradation products. Hence, the author
has developed a stability-indicating HPLC method for quantitative determination of
DOX in pharmaceuticals, and validated the method in accordance with ICH guidelines
[47]. The developed method was applied to the determination of DOX in spiked human
urine. The details of the present titrimetric, spectrophotometric and HPLC methods are
presented in this chapter.
77
SECTION 3.1
NON-AQUEOUS TITRIMETRIC ASSAY OF DOXYCYCLINE HYCLATE IN
PHARMACEUTICAL PREPARATIONS
3.1.1.0 INTRODUCTION
Nonaqueous titration is the titration of substances dissolved in nonaqueous
solvents. It is the most common titrimetric procedure used in 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. The
most commonly used procedure is the titration of organic bases with perchloric acid in
anhydrous acetic acid [48].
Since, water behaves as both a weak acid and a weak base; in an aqueous
environment, it can compete effectively with very weak acids and bases with regard to
proton donation and acceptance, as shown below:
H2O + H+ H3O+
Competes with RNH2 + H+ RNH3+
Substances which are either too weakly basic or too weakly acidic to give sharp
endpoints in aqueous solution can often be titrated in nonaqueous solvents. The non-
aqueous acid base titrations can be explained by means of the concepts of the Brønsted-
Lowry theory. According to this theory an acid is a proton donor, i.e. a substance
which tends to dissociate to yield a proton, and a base is proton acceptor, i.e. a
substance which tends to combine with a proton. When an acid HB dissociates it yields
a proton together with the conjugate base B of the acid:
HB H+ + B -
acid proton base
Many pharmaceutical compounds [49-54] containing primary, secondary and
tertiary amines have been determined through this reaction. Solution of HClO4 in either
78
glacial acetic acid or dioxane solution is frequently used as titrant for titration of weak
bases [49, 55]. Glacial acetic acid, an amphiprotic solvent is widely used for the
titration of weak bases such as amines.
From the literature survey presented in the Section 3.0.2.0, it is observed that no
titrimetric procedure has ever been reported for the determination of DOX in
pharmaceuticals. In this section, the author describes a titrimetric method of DOX
based on non-aqueous acid base reaction. DOX in acetic acid medium was titrated
against acetous perchloric acid with both visual end point detection using crystal violet
as indicator and potentiometric end point detection employing modified glass
electrode-saturated calomel electrode. The methods were successfully applied to the
formulations containing DOX and the results were highly encouraging.
3.1.2.0 EXPERIMENTAL
3.1.2.1 Apparatus
A Metrohm Swiss made Tiamo 809 and 803 potentiometer provided with a
combined glass-SCE electrode system was used for potentiometric titration. The KCl
of the salt bridge was replaced with saturated solution of KCl in glacial acetic acid.
3.1.2.2 Reagents and solutions
All chemicals used were of analytical reagent grade. All solutions are made in
glacial acetic acid (S. D. Fine Chem, Mumbai, India) unless mentioned otherwise.
Perchloric Acid ( 0.01 M): 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 0.01 M perchloric acid and standardized with pure potassium
hydrogen phthalate and crystal violet as indicator [55].
Crystal violet indicator (0.1 %): Prepared by dissolving 50 mg of dye (S. D. Fine
Chem, Mumbai, India) in 50 ml of glacial acetic acid.
Mercuric acetate solution (5 %): Five gram of the pure Hg(OAc)2 (Merck India
ltd, Mumbai, India ) was dissolved in 100 ml of glacial acetic acid, filtered and used.
Standard solution of DOX
Pure DOX (pharmaceutical grade) sample was kindly provided by Lotus
Pharma Ltd, Bangalore, India. Stock standard solution containing 4 mg ml-1
drug was
79
prepared by dissolving the 400 mg of DOX in 100 ml glacial acetic acid in calibrated
flask.
Two brands of tablets, namely, DOX-100 (Dr. Reddy’s Lab) and Doxy-100
(Micro Labs Ltd) were used in the investigation.
3.1.3.0 ASSAY PROCEDURES
3.1.3.1 Visual titration (Method A)
An aliquot of the drug solution containing 4.0-40.0 mg of DOX was measured
accurately and transferred into a clean and dry 100 ml titration flask and the total
volume was brought to 10 ml with glacial acetic acid. Then, 2 ml of 5 % Hg(OAc)2
was added, the content was mixed and after 2 min, two drops of crystal violet indicator
were added and titrated with standard 0.01 M perchloric acid to a blue colour end
point.
A blank titration was performed in the same manner without DOX, and the
necessary volume corrections were made.
The amount of the drug in the measured aliquot was calculated from the
formula:
n
RVMmgAmount w=)(
where V = volume of perchloric acid required, ml; Mw = relative molecular mass of the
drug; and R = molarity of the perchloric acid and n = number of moles of perchloric
acid reacting with each mole of DOX.
3.1.3.2 Potentiometric titration (Method B)
An aliquot of the standard drug solution equivalent to 4.0-40.0 mg of DOX 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 followed by the addition of
2 ml of 5 % Hg(OAc)2. The combined glass-SCE (modified) system was dipped in the
solution. The contents were stirred magnetically and the titrant (0.01 M HClO4) was
added from a microburette. Near the equivalence point, titrant was added in 0.05 ml
increments. After each addition of titrant, the solution was stirred magnetically for 30 s
and the steady potential was noted. The addition of titrant was continued until there
was no significant change in potential on further addition of titrant. The equivalence
80
point was determined by applying the graphical method. The amount of the drug in the
measured aliquot was calculated as described under visual titration.
3.1.3.3 Procedure for tablets
Twenty tablets were weighed and ground into a fine powder. An amount of
powder equivalent to 400 mg DOX was weighed accurately and transferred into a 250
ml round bottomed (RB) flask and sonicated for 5 min with 100 ml of methanol. The
solution was filtered through Whatmann No. 42 filter paper and the filtrate was
collected in a 250 ml RB flask. Then, methanol was evaporated at 40 – 45o
C under the
stream of nitrogen. The resulting residue was dissolved in glacial acetic acid and
transferred into 100 ml standard flask and the volume was brought to 100 ml with
glacial acetic acid. A suitable aliquot was next subjected to analysis by applying the
general procedures as described earlier.
3.1.4.0 RESULTS AND DISCUSSION
3.1.4.1 Chemistry
The reaction between DOX and HClO4 in acetic acid is an acid-base reaction
where the strong acid can donate a proton to nitrogen of the amino group of the drug
molecule [49].
In the presence of perchloric acid, acetic acid will accept a proton:
CH3COOH + HClO
4 CH3COOH2+ + ClO4
-
Basic-N + CH3COOH ⇌ Basic-NH+ + CH3COO
-
2CH3COOH2+ + 2CH3COO- 4CH3COOH
Adding HClO4 + Basic-N ⇌ Basic-NH+ + ClO4
-
The CH3COOH2+ can very readily give up its proton to react with a base, so basic
properties of a base are enhanced and hence, titration between weak base and
perchloric acid can often be accurately carried out using acetic acid as solvent.
DOX is a hydrochloride, which is too weakly basic to react quantitatively with
acetous perchloric acid. Addition of mercuric acetate (which is undissociated in acetic
acid solution) to a halide salt replaces the halide ion by an equivalent quantity of
acetate ion, which is a strong base in acetic acid as shown in the Figure 3.1.1 given
below:
81
Cl2
Cl2 (CH3COO)2Hg HClO42 HgCl2
O
OH
CONH2
NOH
O OHOH
H H
OH
H Cl
O
OH
CONH2
N
H+
OH
O OHOH
H H
OH
+
DOXDOX+
+ +2DOX+2DOX+. 2ClO4
- + 2CH3COOH+
(undissociated)(undissociated)
+
½ C2H6O, ½ H2O½ C2H6O, ½ H2O22
Figure 3.1.1 Possible way of the neutralization reaction.
The enhanced basicity of DOX in acetic acid medium is due to non-lavelling
effect of acetic acid making determination of DOX easier. The procedures involve the
titration of DOX 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 3.1.2). With both methods of
equivalence point detection, a reaction stoichiometry of 1:1 (drug:titrant) was obtained
which served as the basis for calculation. Using 0.01 M perchloric acid, 4.0-40.0 mg of
DOX was conveniently determined. The relationship between the drug amount and the
titration end point was examined. The linearity between two parameters is apparent
from the correlation coefficients of 0.9965 and 0.9986 obtained by the method of least
squares for visual and potentiometric methods, respectively. From this it is implied that
the reaction between DOX and perchloric acid proceeds stoichiometrically in the ratio
1:1 in the range studied.
Figure 3.1.2 Potentiometric titration curves for 20 mg DOX Vs 0.01 M HClO4.
82
3.1.4.2 Method optimization
In both the methods, the optimum amount of mercuric acetate required was
studied by varying its amount and keeping the drug amount constant followed by the
measurement of the stoichometric amount of drug found in each case. It was found
that, a 2 ml of 5 % Hg(OAc)2 was sufficient for complete replacement of chloride in
drug by acetate and the same amount was fixed through out the investigation. A contact
time of 2 min was essential after the addition of mercury(II) acetate.
3.1.4.3 Method validation
Accuracy and precision
The precision of the methods was evaluated in terms of intermediate precision
(intra-day and inter-day). Three different amounts of DOX within the range of study in
each method were analysed in seven and five replicates in method A and method B,
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 DOX showed that the precision of the methods was good
(Table 3.1.1). The accuracy of the methods was determined by the percent mean
deviation from known concentration, and results are presented in Table 3.1.1.
Table 3.1.1 Results of intra-day and inter-day accuracy and precision study
Method
DOX
taken,
mg
Intra-day accuracy and
precision
Inter-day accuracy and
precision
DOX
found,
mg
% RE % RSD
DOX
found,
mg
% RE % RSD
Visual titrimetry,
(n=7)
8.00 24.0
40.0
8.09 23.99
40.10
1.13 0.04
0.25
1.95 0.56
1.02
8.13 24.30
41.07
1.63 1.25
2.68
2.85 1.25
0.99
Potentiometric
titrimetry (n=5)
8.00
24.0 40.0
8.06
24.09 40.08
0.75
0.38 0.20
0.98
0.99 1.06
8.09
24.25 40.92
1.13
1.04 2.3
2.36
1.20 0.89
RE.relative error, RSD. relative standard deviation
Robustness and ruggedness
The robustness of the methods was evaluated by making small incremental
changes in volume of Hg(OAc)2 and standing time after adding Hg(OAc)2, and the
effect of the changes was studied by recording the volumes of HClO4 required to titrate
83
three different amounts separately. The changes had negligible influence on the results
as revealed by small intermediate precision values expressed as % RSD (≤ 2.09 %).
The results are shown in Table 3.1.2.
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 within 2.36 % whereas the inter-burettes RSD for the same DOX amounts
was less than about 2.63 % suggesting that the developed method were rugged. The
results are shown in Table 3.1.2.
Table 3.1.2 Results of robustness and ruggedness expressed as intermediate precision
(% RSD)
Method
DOX
taken,
mg
Robustness Ruggedness
Change in
ml of
Hg(OAc)2*
Change in
standing
time**
, s
Inter-
analysts
(%RSD),
(n=4)
Inter-
instruments
(%RSD),
(n=4)
Visual
titrimetry
10
20 30
2.09
1.66 1.03
1.98
1.56 0.98
2.36
1.36 1.13
2.63
1.56 1.23
Potentiometric titrimetry
10
20
30
1.56
1.26
0.86
1.86
1.35
0.89
1.86
1.30
1.10
1.85
1.36
1.08 *The volume of Hg(OAc)2 varied were 1.8, 2.0 and 2.2 ml.
**Standing times employed were 90, 120 and 150 s.
Application to tablets
The described titrimetric procedures were successfully applied for the
determination of DOX in its tablets (DOX-100 and DOXY-100). The obtained results
(Table 3.1.3) were statistically compared with the official BP method [1]. The method
consisted that the determination of DOX by liquid chromatography with UV detection.
The obtained results by the proposed methods agreed well with those of reference
method and with the label claim. The excipients present in the tablets interfered in the
assay when acetic acid was used as extraction solvent. In order to overcome the
interference, methanol was used as extraction solvent and evaporated to dryness, the
resulting residue was dissolved in acetic acid and used for the assay by following
general procedure. The results were also compared statistically by a Student’s t-test for
accuracy and by a variance F-test for precision with those of the reference method at 95
% confidence level as summarized in Table 3.1.3. The calculated t-and F-values did not
84
exceed the tabulated values inferring that proposed methods are as accurate and precise
as the reference method.
Table 3.1.3 Results of assay in tablets and statistical comparison with official method
*Average of five determinations.
Tabulated t value at the 95% confidence level is 2.77.
Tabulated F value at the 95% confidence level is 6.39.
Recovery studies
To a fixed amount of drug in tablet (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 3.1.4 show that recoveries were in
the range from 98.56 to 103.6 % indicating that commonly added excipients to tablets
did not interfere in the determination.
Brand
name
Label
claim,
mg/tablet
Found* (Percent of label claim ± SD)
Official
method
Proposed methods
Visual
titrimetry
Potentiometric
titrimetry
DOX 100 100 99.06±1.23
101.3±1.95
t=2.23 F=2.51
100.6±1.06
t=2.13 F=1.35
DOXY 100
100 101.6±0.89
103.6±1.40
t=2.76
F=2.47
102.3±0.86
t=1.26
F=1.07
85
Table 3.1.4 Results of recovery study by standard addition method
Visual titrimetry Potentiometric titrimetry
Tablet
studied
DOX
in
tablet,
mg
Pure
DOX
added,
mg
Total
DOX
found,
mg
Pure DOX
recovered
(Percent±SD*)
DOX in
tablet,
mg
Pure
DOX
added,
mg
Total
DOX
found,
mg
Pure DOX
recovered
(Percent±SD*)
DOX
100
10.13
10.13
10.13
5.0
10.0
15.0
15.07
20.29
25.19
98.76±1.36
101.6±1.30
100.4±0.99
10.06
10.06
10.06
5.0
10.0
15.0
15.07
20.02
24.84
100.16±1.23
99.56±1.53
98.56±0.56
DOXY
100
10.36
10.36
10.36
5.0
10.0
15.0
15.54
20.54
25.36
103.6±2.56
101.8±1.89
99.98±0.56
10.23
10.23
10.23
5.0
10.0
15.0
15.35
20.59
25.37
102.3±1.66
103.6±2.6
100.9±1.50 *
Mean value of three determinations.
86
SECTION 3.2
SENSITIVE AND SELECTIVE SPECTROPHOTOMETRIC ASSAY OF
DOXYCYCLINE HYCLATE IN PHARMACEUTICALS USING FOLIN-
CIOCALTEU REAGENT
3.2.1.0 INTRODUCTION
The Folin–Ciocalteu reagent (FC Reagent) or Folin's phenol reagent or Folin–
Denis reagent, also called the Gallic Acid Equivalence method (GAE), is a mixture of
phosphomolybdate and phosphotungstate used for the colorimetric assay of phenolic
and polyphenolic antioxidants [56]. This reagent does not only measure total phenols
and will react with any reducing substance. The reagent therefore measures the total
reducing capacity of a sample, not just the level of phenolic compounds. This reagent
also reacts with some nitrogen-containing compounds such as hydroxylamine and
guanidine [57]. The underlying chemistry for its application in spectrophotometry is that
when F-C reagent gets reduced in the presence of reducing agents like phenols and amine
in alkaline medium, it forms intense blue colored chromogen. This is widely used for the
colorimetric assay of phenolic and polyphenolic antioxidants [56].
F-C reagent is specially used for the determination of many phenolic and amino
compounds utilizing its liability to be reduced into blue colored product. Many drug
substances such as salbutamol [58], minocycline [59], diclofenac [60], rimetazidine
[61], acyclovir [62], methotrexate [63], omeprazole [64], sulphinpyrazone [65], and
gliclazide [66], isoxsuprine hydrochloride [67], diacerein [68], Abacavir sulphate [69],
rizatriptan benzoate [70], granisetron HCl [71] have been determined on this basis.
From the literature survey presented in Section 3.0.2.2, it is observed that DOX
has the capacity to undergo oxidation. Since, FC-reagent reacts with any reducing
substance and no report has ever been found on the use of FC reagent in the
determination of DOX, the author has developed a spectrophotometric method DOX
employing FC reagent. The method is based on the formation of blue colored
chromogen due to reduction of tungstate and/or molybdate in Folin-Ciocalteu (F-C)
reagent by DOX in alkaline medium. The colored species has an absorption maximum
at 770 nm. The details of the method are presented in this section 3.2.
87
3.2.2.0 EXPERIMENTAL
3.2.2.1 Apparatus
The instrument is the same that was described in Section 2.1.2.1.
3.2.2.2 Reagents
All chemicals used were of analytical reagent grade and distilled water was used
throughout the study.
F-C reagent (1 N): An aqueous solution of 1 M (1:1 v/v) of the reagent was prepared
by diluting 50 ml of the commercially available 2 N F-C reagent to 100 ml in a
standard flask.
Na2CO3 (20%): Prepared by dissolving 20 g of the chemical (S.D. Fine Chem Ltd,
India) in 100 ml of water.
Preparation of standard DOX solution
A stock standard solution of DOX (300 µg ml-1
) was prepared by dissolving
pure DOX in water in a standard flask. Working concentration of DOX (30 µg ml-1
)
was prepared by dilution of the above stock solution with water.
Tablets used were the same mentioned in section 3.1.1.2 In addition, Microdox-
DT ( Micro Labs Ltd, Bangalore, India) was also used
3.2.3.0 ASSAY PROCEDURE
3.2.3.1 Procedure for calibration graph
Different aliquots of working standard DOX solution (30 µg ml-1
) ranging from
0.0-4.0 ml were transferred into a series of 10-ml of standard flasks and the total
volume was brought to 4 ml with water. To each flask, 3 ml of 20% Na2CO3 and 2 ml
of F-C reagent (1 N) solution were successively added by means of a microburette. The
flasks were stoppered, contents were mixed well and kept to room temperature for 20
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 but in the
absence of DOX.
Calibration graph was prepared by plotting the increasing absorbance values
versus concentrations of DOX. The concentration of the unknown was read from the
respective calibration graph or deduced from the regression equation derived using the
Beer’s law data.
88
3.2.3.2 Procedure for tablets
An amount of finely ground tablet powder equivalent to 3 mg of DOX was
accurately weighed into a 100-ml standard flask, the flask was shaken after addition of
a 70 ml of water for about 20 min and finally volume was made upto the mark with
water. The content was kept aside for 5 min, and filtered using Whatman No. 42 filter
paper. First 10-ml portion of the filtrate was discarded and a suitable aliquot was used
for assay as described under Section 3.2.3.1.
3.2.3.3 Placebo blank and synthetic mixture analysis
A placebo blank containing talc (25 mg), starch (30 mg), lactose (30 mg),
calcium carbonate (5 mg), calcium dihydrogen orthophosphate (10 mg), methyl
cellulose (20 mg), sodium alginate (30 mg) and magnesium stearate (20 mg) was
prepared; 20 mg was extracted with water and solution made as described under
Section 3.2.3.2 and then subjected to analysis using the procedure described above.
A synthetic mixture was prepared by adding 20 mg of DOX to about 20 mg
placebo blank prepared above, homogenized and the solution was prepared as done
under Section 3.2.3.2. The filtrate was collected in a 100-ml flask and the resulting
synthetic mixture solution (200 µg ml-1
in DOX) was appropriately diluted to get 30 µg
ml–1
solution, and subjected to analysis.
3.2.4.0 RESULTS AND DISCUSSION
The structural features of DOX allowed the use of F-C reagent for its assay.
The proposed method is based on the formation of a blue colored chromogen when
DOX reacted with the F-C reagent in the presence of sodium carbonate. The colour
formation may be explained as follows based on analogy reported by Peterson [68].
The mixed acids in the F-C reagent involve the following chemical species:
3H2O·P2O5·13WO3·5MoO3·10H2O and 3H2O·P2O5·14WO3·4MoO3·10H2O
DOX probably effects a reduction of 1,2 or 3 oxygen atoms from tungstate and/or
molybdate in the F-C reagent, there by producing one or more of the reduced species
which have characteristic intense blue color.
89
3.2.4.1. Method optimization
Optimum conditions were fixed by varying one parameter at a time while
keeping other parameters constant and observing its effect on the absorbance at 770
nm.
Absorption spectra
DOX reacts with F-C reagent in the presence of Na2CO3 to form intensely blue
coloured product with an absorption maximum at 770 nm. Fig 3.2.1 shows the
absorption spectra of the reaction product and reagent blank. Under the same
experimental conditions the blank had negligible absorbance.
a
b
Figure 3.2.1 Absorption spectra of: a) reaction product of DOX (8.0 µg ml-1
) with F-C
reagent in Na2CO3 solution and b) blank
Selection of reaction medium
To find a suitable medium for the reaction, different aqueous bases such as
sodium hydroxide, sodium carbonate or bicarbonate, sodium acetate and sodium
hydrogen phosphate were investigated. Best results were obtained with sodium
carbonate. It was found that maximum and constant absorbance values were obtained
in the concentration range of 0.28 – 0.57 M Na2CO3 thus 0.43 M was fixed as
optimum.
Effect of F-C reagent concentration
Several experiments were carried out to study the influence of F–C reagent
concentration on the color development. It is apparent that 0.5 – 0.2 M of reagent gave
the maximum color intensity, thus 0.2 M of reagent was used throughout the
investigation.
90
Reaction time
The reaction time was studied by measuring the absorbance of the blue
chromogen after mixing the reactants in the time ranging from 2 min to 2 h. Maximum
color was developed in 20 min, and the color was stable for at least 60 min thereafter.
Therefore, measurements were made only after 20 min throughout the investigation.
Order of the addition of the reactants
The effect of the order of addition was studied by measuring the absorbance of
the colored systems by adding the reactants in different order. Greatest sensitivity was
achieved when the order was maintained as described in the procedure and the same
was followed throughout the investigation.
3.2.4.2 Method validation
Linearity
A linear correlation was found between absorbance at λmax and concentration of
DOX in the range 0.75 – 12 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). A plot of log absorbance versus log concentration yielded a
straight line with slope equal to 0.9922 further establishing the linear relation between
the two variables. The optical characteristics such as Beer’s law limits and molar
absorptivity values of the method are given in Table 3.2.1.
Table 3.2.1. Sensitivity and regression parameters
Parameter Value
λ, nm 770
Linear range, µg ml-1
0.75-12.0
Molar absorptivity(ε), l mol-1
cm-1
2.78 ×104
Limit of detection (LOD), µg ml-1
0.08
Limit of quantification (LOQ), µg ml-1
0.20
Regression equation, Y = aX+b:
intercept (a) 0.0054±0.0051
slope (b) 0.0531±0.0006
Regression coefficient (R) 0.9997
Limit of determination as the weight in µg per mL 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, a is intercept, b is slope.
91
Accuracy and precision
The intra-day and inter-day precision was evaluated by measuring the
absorbance of seven replicate samples at 3.0, 6.0 and 9.0 µg ml-1
concentration levels
of DOX. The percentage relative standard deviation (%RSD) values were ≤ 1.6%
(intra-day) and ≤ 2.1% (inter-day) indicating high precision of the method. Percent
relative error (eR) values of ≤ 2.7% demonstrate the high accuracy of the proposed
method. The results are summarized in Table 3.2.2.
%RE. Percent relative error, %RSD. relative standard deviation
Selectivity
In the analysis of placebo blank solution the absorbance in each case was equal
to the absorbance of the blank which revealed no interference. To assess the role of the
inactive ingredients on the assay of DOX, the general procedure was followed by
taking 4, 6 and 8 µg ml-1
DOX solution prepared by using synthetic mixture. The
recovery values obtained from this study are presented in the Table 3.2.3. The values
97.4 – 104.3% with RSD values of <3% clearly indicates the non-interference of the
inactive ingredients in the assay of DOX.
Table 3.2.3 Results of recovery of DOX from synthetic mixture analysis
DOX taken
(µg ml-1
)
DOX found
(µg ml-1
)
DOX
recovered (%)*
4.00
6.00
8.00
3.93
5.84
6.26
98.3±1.28
97.4±2.21
104.3±2.95 *±SD, n = 3
Table 3.2.2 Results of intra-day and inter-day accuracy and precision study
DOX
taken
(µg ml-1
)
Intra-day accuracy and
precision
(n=7)
Inter-day accuracy and precision
(n=5)
DOX
found,
(µg ml-1
)
RE
(%)
RSD
(%)
DOX found,
(µg ml-1
)
RE
(%)
RSD
(%)
3.0
6.0
9.0
2.95
6.06
9.08
1.7
1.0
0.9
1.6
1.0
0.9
3.08
6.10
9.12
2.7
1.7
1.3
2.1
2.5
2.0
92
Robustness and ruggedness
The robustness of the methods was evaluated by making small incremental
changes in the volumes of Na2CO3 and reaction time, 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 values
expressed as RSD (≤ 1.89%). In order to check the ruggedness of the method, the assay
was performed using the same operational conditions but using different cuvettes in
two different laboratories, different analysts and different elapsed time. Results
obtained from inter-lab, inter-day and inter-analysts were reproducible. The inter-
analysts RSD were within 2.5% whereas the inter-cuvettes and inter-lab RSD for the
same DOX amount was less than about 2.6% suggesting that the developed method
was rugged. The results of this study are presented in the Table 3.2.4.
Table 3.2.4 Results of robustness and ruggedness expressed as intermediate
precision (% RSD)
DOX
taken,
µg ml-1
Robustness Ruggedness
Parameters altered Inter-labs,
(%RSD)
(n=4
Inter-
analysts
% RSD
(n=4)
Inter-
cuvettes
% RSD
(n=4)
Volume of
reactants*
Reaction
timeΨ
3.0
9.5
12.0
1.58
1.13
1.52
1.89
1.54
1.70
1.56
2.11
2.52
2.10
1.99
2.49
2.52
2.33
2.59 *The volumes reactant was 3±0.2 ml of Na2CO3 and. ΨThe reaction times were 20±1 min,
respectively.
Application to tablets
Method was applied to the determination of DOX in three brands of tablets.
The results obtained were statistically compared with the official BP method [1]. The
results obtained by the proposed method agreed well with those of reference method.
The results were also compared statistically by Student’s t-test for accuracy and by a
variance F-test for precision with those of the reference method at 95 % confidence
level. The results showed that the calculated t-and F-values did not exceed the
tabulated values inferring that proposed method is as accurate and as precise as the
reference method. The results are shown in Table 3.2.5.
93
Recovery studies
The test was done by spiking the pre-analyzed tablet powder with pure DOX at
three different levels and the total was found by the proposed method. Each test was
repeated three times. In all the cases, the recovery percentage values ranged between
98.9 and 103.6% with relative standard deviation in the range 0.56-1.35%. In all the
cases, closeness of the results to 100% (Table 3.2.6) showed the fairly good accuracy
of the method.
Table 3.2.6 Results of recovery study using standard addition method
Tablet
studied
DOX in tablet
extract,
µg ml-1
Pure DOX
added,
µg ml-1
Total
DOX
found,
µg ml-1
Pure DOX
recovered
(Percent ± SD*)
DOX 100
4.16
4.16
4.16
2.50
5.00
7.50
6.63
9.17
11.83
98.9±1.0
100.1±0.9
102.3±1.2
DOXY 100
4.16
4.16
4.16
2.50
5.00
7.50
6.65
9.22
11.93
99.6±0.6
101.2±1.3
103.6±1.4 *Mean value of three determinations.
Table 3.2.5 Results of analysis of tablets by the proposed method and statistical
comparison with the reference method
Tablet
brand
name
DOX
taken,
µg ml-1
Found± SD*
Student’s
t- value
Variance
ratio F-
value Reference
method
Proposed
method
DOX-T 100
6.00
9.00
5.99±0.03
8.94±0.07
5.97±0.03
9.01±0.04
1.05
2.01
1.00
3.06
DOXY 100 6.00
9.00
5.97±0.03
8.96±0.05
5.96±0.03
9.05±0.06
0.52
2.59
1.00
1.44
Micrdox-DT
100
6.00
9.00
5.97±0.02
9.03±0.04
5.94±0.04
8.94±0.07
1.58
2.59
4.00
3.06 *Average of five determinations.
Tabulated t value at the 95% confidence level is 2.77. Tabulated F value at the 95%
confidence level is 6.39.
94
SECTION 3.3
TITRIMETRIC AND SPECTROPHOTOMETRIC DETERMINATION OF
DOXYCYCLINE HYCLATE USING BROMATE-BROMIDE, METHYL
ORANGE AND INDIGO CARMINE
3.3.1.0 INTRODUCTION
An acidified mixture of bromate and bromide actually behaves as an equivalent
solution of bromine. In acidic medium, potassium bromate is a strong oxidizing agent
with a standard redox potential of 1.52 V.
In acid solution, potassium bromated is reduced smoothly to bromide and then
reacts with excess bromate to yield free bromine.
BrO3- + 6H+ + 6e- Br- + 3H2O
BrO3- + 5 Br- + 6H+
3Br2 + 3H2O
It is usual to add bromide to the test solution before the titration or to include
in the standard bromate solution so that only the second reaction is involved. Bromate-
bromide mixture is an eco-friendly green brominating agent [73]. Thus the stable
bromate-bromide solution serves for the extemporaneous preparation of a standard
solution of bromine. Aqueous bromine solutions are unstable because of high vapour
pressure of bromine. And also bromine vapors are very toxic with inhalation [74].
Bromate–bromide mixture in acid medium has been extensively used for the
direct titrimetric assay of wide-ranging pharmaceuticals [75-77], with visual,
electrometric or photometric detection of end point. Some of the examples reported
include salbutamol sulphate [78], captopril [79], ranitidine [80], adrenergic drugs [81],
amethoclain hydrochloride [82], oxyphenbutazone [83], phenolic steroids [84],
isonicotinic acid hyrazide [85], ledol [86], tuberulostatic drugs [87], and nizatidine
[88], ascorbic acid [89-91], aminosalicylic acid [92], citral [93], thiamine
hydrochloride [94, 95], cimetidine [96], secobarbital [97], carbimazole [98],
albendazole [99] sulphonamide [100], atenolol [101, 102], carbamazepine [103],
domperidone [104], simvastatin [105] and stavudine [106].
Methyl orange and indigo carmine are irreversibly bleached by insitu generated
bromine [75], and the bleaching action has successfully been utilised for the indirect
spectrophotometric assay of a wide ranging pharmaceuticals such as albendazole [99],
95
salbutamol sulphate [78], captopril [79], ranitidine [80], famotidine [107],
chlorpromazine [108], astemizole [109], prochlorperazine [110] mebrophenhydramine
[111], felodipine [112], amoxycillin [113] trifluoperazine [114], frusemide [115],
cyproheptadine [116], metaprolol tartrate [117] and amlodipine besylate [118].
From the literature survey presented in Section 3.0.2.0 and from the foregoing
paragraphs, it is clear that bromate-bromide reagent has not been used for the assay of
DOX in pharmaceuticals. In this section, one titrimetric and two indirect
spectrophotometric methods are described for the determination of DOX in bulk drug
and in its tablets using bromate-bromide, methyl orange and indigo carmine as
reagents. In titrimetry (method A), DOX is treated with a known excess of bromate-
bromide mixture in acid medium and the residual bromine is back titrated
iodometrically after the reaction between DOX and in situ bromine is ensured to be
complete. In spectrophotometric methods, the excess of bromine is estimated by
treating with a fixed amount of either methyl orange (method B) or indigo carmine
(method C) and measuring the change in absorbance either at 520 or 610 nm. The
details are presented in this section.
3.3.2.0 EXPERIMENTAL
3.3.2.1 Apparatus
The instrument is the same that was described in Section 2.1.2.1.
3.3.2.1 Reagents
All chemicals used were of analytical reagent grade and distilled water was used
throughout the study.
Bromate-Bromide mixture: A bromate-bromide solution equivalent to 5mM KBrO3-
50 mM KBr was prepared by dissolving accurately weighed 418 mg of KBrO3 (S.d.
Fine Chem Ltd, Mumbai, India) and 3 g of KBr (Merck, Mumbai, India) in water and
diluting to the mark in a 500 ml calibrated flask and this solution was used in
titrimetric work. For use in spectrophotometric study, a 1000 µg ml-1
KBrO3 solution
containing a large excess of KBr was prepared by dissolving 100 mg of KBrO3 and 1 g
of KBr in water and diluting to the mark in a 100 ml calibrated flask. This was diluted
stepwise to get 10 µg ml-1
and 30 µg ml-1
bromate solutions for use in method B and
method C, respectively.
96
Sodium thiosulphate: A 0.03 M sodium thiosulphate (S.d. Fine Chem Ltd, Mumbai,
India) solution was prepared in water and standardized [119].
Potassium iodide: A 10% aqueous solution of KI (LobaChemie, Mumbai, India) was
prepared by dissolving 10g of the chemical in 100 ml of water.
Starch indicator: To prepare 1 % starch indicator, 1 g of the chemical (Merck,
Mumbai, India) was made into a paste with water and poured into 100 ml of boiling
water, boiled for 1 min, cooled and used in method B (titrimetry).
Hydrochloric acid: Concentrated hydrochloric acid (Merck, Mumbai, India; Sp. gr.
1.18) was diluted appropriately with water to get 2 and 5 M HCl.
Methyl orange (50 µg ml-1
) and indigo carmine indicator (200 µg ml-1
): The
indicators were prepared by dissolving 5.9 mg of methyl orange (S. D. Fine Chem.
Ltd., Mumbai, India; dye content 85 %) and 22.2 mg of indigo carmine (S.d. Fine
Chem, Mumbai, India, dye content 90%) in 100 ml water.
Preparation of standard DOX solution
A 1 mg ml-1
standard drug solution was prepared by dissolving 250 mg of DOX
in water, the volume was made upto 250 ml in a calibrated flask with water and was
used in titrimetry. This solution was then diluted stepwise with water to get 5 µg ml-1
and 10 µg ml-1
solutions for use in method B and method C, respectively.
Tablets used were same as mentioned in section 3.1.
3.3.3.0 ASSAY PROCEDURES
3.3.3.1 Method A (Titrimetry)
An aliquot of pure drug solution containing 1-8 mg of DOX was transferred
accurately into a 100 ml Erlenmeyer flask and the total volume was made upto 10 ml
with water. The solution was acidified by adding 5 ml of 2 M HCl. Ten ml of bromate-
bromide solution (5 mM w.r.t KBrO3) was transferred to the flask by means of a
pipette. The flask was stoppered, the content mixed well and kept aside for 20 min with
occasional swirling. The stopper was then washed with 5 ml of water and 5 ml of 10%
potassium iodide solution was added to the flask. The liberated iodine was titrated with
0.03 M sodium thiosulphate to a starch end point. A blank titration was run under
identical conditions and the amount of drug in the measured aliquot was calculated
from:
97
n
VMwRmg =
where V = volume of bromate reacted; Mw = relative molecular mass of drug; R =
molar concentration of bromate; n = number of moles of bromate reacting with each
mole of drug.
3.3.3.2 Method B (Spectrophotometry using methyl orange)
Different aliquots (0.0-2.5 ml) of 5 µg ml-1
DOX solution were accurately
measured into a series of 10 ml calibrated flasks and the total volume was adjusted to
2.5 ml with water. To each flask were added 1 ml each of bromate-bromide solution
(10 µg ml-1
w. r. t. KBrO3) and 5 M hydrochloric acid. The content was mixed well and
let stand for 20 min with occasional shaking. Then 1 ml of 50 µg ml-1
methyl orange
solution was added to each flask and diluted to the mark with water. The absorbance of
each solution was measured at 520 nm against a reagent blank after 5 min.
3.3.3.3. Method C (Spectrophotometry using indigo carmine)
Varying aliquots of standard DOX solution (0.0-5.0 ml) of 10 µg ml-1
DOX
were transferred into a series of 10 ml calibrated flasks by means of a micro burette,
and the total volume was brought to 5 ml by adding water. To each flask, 1 ml of 5 M
HCl and 1.5 ml of bromate-bromide solution (30 µg ml-1
w.r.t. KBrO3) were added.
After mixing the content, the flasks were allowed to stand for 10 min with occasional
shaking. Then, 1 ml of 200 µg ml-1
indigo carmine solution was added to each flask
and diluted to the mark with water. The absorbance was measured at 610 nm against a
reagent blank after 10 min.
In method B and method C, a calibration graph was prepared by plotting
absorbance versus concentration of drug and the concentration of the unknown was
read from the calibration graph or computed from the regression equation derived from
the Beer’s law data.
3.3.3.4 Procedure for tablets
Twenty tablets each containing 100 mg of DOX were weighed accurately and
pulverized. An amount of tablet powder equivalent to 100 mg was transferred into a
100 ml standard flask. The content was shaken well with about 70 ml of water for 20
min. The mixture was diluted to the mark with water. It was filtered using Whatmann
98
No 42 filter paper. First 10 ml portion of the filtrate was discarded and a 5 ml aliquot
was subjected to analysis following the procedure described in method A. For method
B and method C, the tablet solution (1000 µg ml-1
in DOX) was diluted appropriately
with water to get 5 and 10 µg ml-1
DOX and suitable portions were used in the analysis
by following the general spectrophotometric procedures described for pure drug.
3.3.3.5 Placebo blank and synthetic mixture analysis
Twenty mg of placebo blank prepared as described in section 3.2 was extracted
with water and solution made as described under “section 3.3.3.4”. A convenient
aliquot of solution was subjected to analysis by titrimetry (method A) and
spectrophotometry (method B and method C) according to the recommended
procedures.
A synthetic mixture was prepared by adding 100 mg of DOX to about 100 mg
placebo blank prepared above, homogenized and the solution was prepared as done
under ‘section 3.3.3.4’ and a 5 ml aliquot was assayed by method A. The synthetic
mixture solution (1000 µg ml-1
in DOX) was appropriately diluted to get 5 and 10 µg
ml–1
solutions, and appropriate aliquots were subjected to analysis by method B and
method C, separately.
3.3.4.0 RESULTS AND DISCUSSION
The determination of DOX is based on oxidation and bromination reaction by
bromine generated in situ by the action of acid on bromate-bromide mixture. In
titrimetry, the reaction is followed by back titration of the residual bromine
iodometrically and in spectrophotometry it is followed by change in absorbance of red
colour of methyl orange at 520 nm (Figure 3.3.1) or blue colour of indigo carmine at
610 nm (Figure 3.3.2), the change being caused by the bleaching action of bromine on
the dyes. In titrimetry (method A) the stoichiometry was expressed as the number of
moles of bromate reacting with each mole of the drug. The reaction stoichiometry was
found to be 1:2 (DOX: KBrO3) in the range of 1-8 mg DOX. Outside this range non-
stoichiometric results were obtained. Since two moles of bromate (equivalent to 12
moles of bromine) are consumed in the reaction, two moles of bromine are believed to
have been used up for the oxidation of the phenolic-OH groups at 5th and 12
th positions
of tetracene, two for the bromination at 7th and 8
th positions, one mole each of bromine
99
is likely to have been used up in the bromination of the amide group [120] as well as
oxidation of ethanolic moiety. The probable reaction scheme is shown in Figure 3.3.3.
0
0.2
0.4
0.6
0.8
400 440 480 520 560 600 640 680 720Wavelength, nm
Ab
so
rban
ce
C
B
A
Figure 3.3.1 Absorption spectra of methyl orange in the presence of A:0.25 µg ml-1
DOX; B: 0.75 µg ml-1
DOX and C: 1.25 µg ml-1
DOX.
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
400 440 480 520 560 600 640 680 720
Wavelength, nm
Ab
so
rban
ce
c
b
a
Figure 3.3.2 Absorption spectra of indigo carmine in the presence of a:1.0 µg ml-1
DOX; b: 3.0 µg ml-1
DOX and c: 5.0 µg ml-1
DOX.
O
OH
NOH
O OHOH
H H
OHO
NH2
H Cl
OH O
O
O
NO
O OOH
H
OHO
NH
Br
Br Br
H Cl
12Br2+
H2O H+
+ 18HBr
Oxidized and brominated product of DOX
H2O
2 2
Unreacted Br2
Method A Determined by iodometric titration
Unreacted Br2
Method B
Method C
+
+
fixed amount of methyl orange
fixed amount of indigo carmine
H+
H+
Absorbance measured at 520 nm
Absorbance measured
at 610 nm
Figure 3.3.3 Probable reaction scheme showing the oxidation and bromination of
DOX, and determination of in situ generated bromine by titrimetry and
spectrophotometric methods.
100
3.3.4.1 Optimization of reaction conditions
Titrimetry
The reaction stoichiometry was found to be unaffected in the presence of 3-8 ml
of 2 M HCl in a total volume of 23-27 ml, and 5 ml was chosen as the optimum
volume and better results and consistent stoichiometry were obtained in the preferred
HCl medium than the other acid media studied (H2SO4, H3PO4 and CH3COOH). The
reaction was found to be complete in 15 min and contact time up to 30 min had no
effect on the stoichiometry or the results. A 10 ml volume of 5 mM bromate solution in
the presence of a large amount of bromide was found adequate for quantitative reaction
with DOX in the range investigated.
Spectrophotometry
Many dyes are irreversibly destroyed to colourless products by oxidizing agents
in acid medium [50] and this observation has been exploited for the indirect
spectrophotometric determination of some bioactive compounds [121-130]. In the
proposed spectrophotometric methods, the ability of bromine to cause bromination and
oxidation of DOX and irreversibly destroy methyl orange and indigo carmine dyes to
colourless products in acid medium has been used. Both spectrophotometric methods
are based on the bromination and oxidation of DOX by a measured excess of in situ
generated bromine and subsequent determination of the unreacted bromine by treating
with methyl orange or indigo carmine and measuring the absorbance at 520 nm
(Figure 3.3.1) or 610 nm (Figure 3.3.2). In either method, the absorbance increased
linearly with increasing concentration of DOX (Figure 3.3.4).
0.00
0.10
0.20
0.30
0.40
0.50
0.60
0.70
0.00 0.25 0.50 0.75 1.00 1.25 1.50
Concentration of DOX (µg mL-1)
Ab
so
rban
ce
0.00
0.20
0.40
0.60
0.80
0.00 1.00 2.00 3.00 4.00 5.00
Concentration of DOX (µg mL-1)
Ab
so
rba
nc
e
Meth
od B
Method C
Figure 3.3.4 Calibration curves
101
DOX, when added in increasing concentrations to a fixed concentration of in
situ generated bromine, consumes the latter proportionately and there will be a
concomitant decrease in its concentration. When a fixed concentration of either dye is
added to decreasing concentrations of bromine, a concomitant increase in the
concentration of dye is obtained. This is observed as a proportional increase in
absorbance at the respective λmax with increasing concentration of DOX (Figures 3.3.1,
3.3.2 and 3.3.4).
Preliminary experiments were performed to fix the upper limits of the dye
concentrations that could be measured spectrophotometrically, and these were found to
be 5 µg ml-1
and 20 µg ml-1
for methyl orange and indigo carmine, respectively. A
bromate concentration of 1.0 µg ml-1
was found to irreversibly destroy the red colour of
5 µg ml-1
methyl orange whereas 4.5 µg ml-1
oxidant was required to bleach the blue
colour due to 20 µg ml-1
indigo carmine in acid medium. Hence, different
concentrations of DOX were reacted with 1.0 ml of 10 µg ml-1
bromate in method B
and 1.5 ml of 30 µg ml-1
oxidant in method C in the presence large excess of bromide
and in acid medium followed by the determination of the residual bromine as described
under the respective procedures.
None of the acids (H2SO4, H3PO4 and CH3COOH) showed precise and accurate
results than HCl. Therefore, HCl was the medium of choice for the bromination and
oxidation of DOX by bromine as well as the latter’s determination employing the dyes.
The absorbance of the dyes was not affected in 0.25 –1.00 and 0.25-1.5 M hydrochloric
acid concentration for method B and method C, respectively. However, since 1 ml of 5
M acid in a total volume of about 5.5 and 8.5 ml for method B and method C,
respectively, was found sufficient to cause bromination and oxidation of drug in a
reasonable time of 20 and 10 min, respectively, the same concentration (0.5 M overall)
was maintained for the determination of unreacted bromine with the dyes. The
specified acid concentration for bromination reaction was not critical. The reaction was
found to be complete in 20 and 10 min for method B and method C, respectively, and
contact times up to 60 min had no effect on the absorbance of the dyes. A contact time
of 5 min (method B) and 10 min (method C) was necessary for the bleaching of the dye
colour by the residual bromine. The absorbance of either dye solution even in the
102
presence of the brominated drug product was found to be stable for more than 48 hours
under these optimized conditions
3.3.4.2 Method validation
Analytical parameters of spectrophotometric methods
A linear correlation was found between absorbance at λmax and concentration of
DOX in the ranges given in Table 3.3.1. 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 3.3.1.
A plot of log absorbance and log concentration, yielded straight lines with slopes equal
to 0.998 and 0.978 for method B and method C, respectively, further establishing the
linear relation between the two variables. The optical characteristics such as Beer’s law
limits, molar absorptivity and Sandell sensitivity values of both methods are also given
in Table 3.3.1. The high values of ε and low values of Sandell sensitivity and LOD
indicate the high sensitivity of the proposed methods.
Table 3.3.1 Sensitivity and regression parameters
Parameter Method B Method C
λmax, nm 520 610
Linear range, µg ml-1
0.125-1.25 0.5-5.0
Molar absorptivity(ε), l mol-1
cm-1
2.62 ×105
6.97 × 104
Sandell sensitivity*, µg cm
-2 0.002 0.010
Limit of detection (LOD), µg ml-1
0.02 0.091
Limit of quantification (LOQ), µg ml-1
0.07 0.27
Regression equation, Y**
Intercept (a) -0.003 -0.001
Slope (b) 0.516 0.14
Standard deviation of a (Sa) 0.0998 0.10
Standard deviation of b (Sb) 0.1331 0.03
Variance (Sa2) 9.96 × 10
-3 0.01
Regression coefficient (r) 0.9997 1.000 *Limit of determination as the weight in µg per ml 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.
Accuracy and precision
To compute the accuracy and precision, the assays described under “general
procedures” were repeated seven times within the day to determine the repeatability
103
(intra-day precision) and five times on five different days to determine the intermediate
precision (inter-day precision) of the methods. These assays were performed for three
levels of analyte. The results of this study are summarized in Table 3.3.2. The
percentage relative standard deviation (% RSD) values were ≤ 2.66% (intra-day) and ≤
2.98% (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 DOX. Bias {bias % = [(Concentration found - known concentration)
x 100 / known concentration]} was calculated at each concentration and these results
are also presented in Table 3.3.2. Percent relative error (%RE) values of ≤ 3%
demonstrate the high accuracy of the proposed methods.
Selectivity
In all the three methods, results of placebo blank and synthetic mixture analyses
revealed that the inactive ingredients used in the preparation had no role in the assay of
active ingredient. To study the role of additives added to the synthetic sample, five ml
of the resulting solution was assayed (n=5) by titrimetry which yielded a % recovery of
98.56 ± 0.98. The synthetic mixture analysis by spectrophotometric methods yielded
percentage recoveries of 97.56 – 103.65 with %RSD values in the range 1.02 – 2.53.
These results demonstrated the accuracy as well as the precision of the proposed
Table 3.3.2 Results of intra-day and inter-day accuracy and precision study
Method DOX
taken*
Intra-day accuracy and precision
(n=7)
Inter-day accuracy and
precision
(n=5)
DOX
Found* ±CL
%RE %RSD DOX
found*±CL
%RE %RSD
A
2.0
4.0
6.0
2.03±0.04
3.93±0.10
6.12±0.14
1.50
1.75
2.00
1.88
2.65
2.45
2.05±0.05
4.05±0.14
6.10±0.23
2.50
1.25
1.67
2.12
2.89
2.98
B
0.50
0.75
1.00
0.51±0.01
0.76±0.01
0.99±0.02
2.00
1.33
1.00
2.66
1.59
2.48
0.49±0.10
0.76±0.01
1.03±0.01
2.00
1.33
3.00
1.50
1.44
0.99
C 2.0 3.0
4.0
2.05±0.04 2.96±0.03
3.94±0.06
2.50 1.33
1.50
2.12 1.26
1.65
2.04±0.04 3.07±0.08
4.08±0.11
2.00 2.33
2.00
1.53 1.98
2.13 .*The values are in mg for method A and µg ml-1 for method B and method C %RE. Percent relative error, %RSD. relative standard deviation and CL. Confidence limits were calculated from: CL = ± tS/√n. (The tabulated value of t is 2.45 and 2.77 for six and four degrees of freedom respectively, at the 95% confidence level; S = standard deviation and n = number of measurements
104
methods and complement the findings of the placebo blank analysis with respect to
selectivity.
Robustness and ruggedness
The robustness of the methods was evaluated by making small incremental
changes in the volume of acid [method A (6.0 mg DOX): 4.0, 5.0 and 6.0 ml; method
B (1.0 µg ml-1
) and method C (3.0 µg ml-1
) : 0.8, 1.0 and 1.2 ml] and contact time
(method A and method B: 19, 20 and 21 min; method C: 9.5, 10.5 and 11.5 min) and
the effect of the changes was studied on the absorbance of the dye colour. The changes
had negligible influence on the results as revealed by small intermediate precision
values expressed as % RSD (≤ 2.68%). Method ruggedness was expressed as the RSD
of the same procedure applied by four different analysts as well as using three different
instruments, (burettes in method A and spectrophotometer in method B and method C).
The inter-analysts RSD were within 2.89% whereas the inter-instruments RSD for the
same DOX concentrations ranged from 1.99-2.89% suggesting that the developed
methods were rugged (Table 3.3.2).
Table 3.3.2 Results of robustness and ruggedness expressed as intermediate
precision (%RSD)
Method
DOM
taken,
mg
Method robustness Method ruggedness
Parameter altered
HCl , ml*
%RSD
(n = 3)
Reaction
time**
,
min %RSD
(n = 3)
Inter-
analysts’
%RSD
(n = 4)
Inter-
burettes’
(n = 3)
A
2.0 0.97 1.14 1.27 1.21
4.0 0.83 1.03 1.61 1.73
6.0 0.75 0.95 1.34 1.99
B
0.50 1.44 1.02 2.15 1.15
0.75 1.17 1.18 2.89 1.56
1.00 1.86 1.89 2.45 1.79
C
2.0 2.68 1.65 1.98 2.89
3.0 2.13 1.98 1.45 2.05
4.0 1.97 1.35 1.87 1.87 *HCl volumes used were 4.0, 5.0 and 6.0 ml of 2 M in method A and 0.8, 1.0 and 1.2 ml of 5 M in method B and method C. **Reaction times altered were 19, 20 and
21 in method A and method B, 9.5, 10.5 and 11.5 min in method C.
105
Application to tablets
The proposed methods were applied to the determination of DOX in two
representative tablets. The results in Table 3.3.4 show that the methods are successful
for the determination of DOX and that the excipients in the dosage forms did not
interfere. The results obtained (Table 3.3.4) were statistically compared with the
official BP method [1]. The results obtained by the proposed methods agreed well with
those of reference method and with the label claim. When the results were statistically
compared with those of the reference method by applying the Student’s t-test for
accuracy and F-test for precision, the calculated Student’s t- value and F-value at 95%
confidence level did not exceed the tabulated values of 2.77 and 6.39, respectively, for
four degrees of freedom. Hence, no significant difference exists between the proposed
methods and the reference method with respect to accuracy and precision.
Recovery studies
To further assess the accuracy of the methods, recovery experiments were
performed by applying the standard-addition technique. The recovery was assessed by
determining the agreement between the measured standard concentration and added
known concentration to the sample. The test was done by spiking the pre-analyzed
tablet powder with pure DOX 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.
Table 3.3.4 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
Proposed methods
Method A Method B Method C
DOX-T
100
100
98.69±0.98
99.12±1.58
t = 0.53
F = 2.60
98.14±1.24
t = 0.78
F = 1.60
97.86±1.3
4
t = 1.13
F = 1.87
DOXY
100
100
101.3±1.14
102.5±1.44
t = 1.47
F = 1.59
100.4±2.44
t = 0.79
F = 4.58
101.8±2.0
4
t = 0.50
F = 3.20 *Average of five determinations.
Tabulated t value at the 95% confidence level is 2.77. Tabulated F value at the 95%
confidence level is 6.39.
106
Each test was repeated three times. In all the cases, the recovery percentage values ranged between 95.87 and 106.3% with
relative standard deviation in the range 1.09 - 2.38%. Closeness of the results to 100 % showed the fairly good accuracy of the
methods. The results are shown in Table 3.3.5.
Table 3.3.5 Results of recovery study by standard addition method
Method A Method B Method C
Tablet
studied
DOX in
tablet
extract,
mg
Pure
DOX
added,
mg
Total
DOX
found,
mg
Pure DOX
recovered
(Percent±SD*)
DOX in
tablet
extract,
µµµµg ml-1
Pure
DOX
added,
µµµµg ml-1
Total
DOX
found,
µµµµg ml-1
Pure DOX
recovered
(Percent±SD*)
DOX in
tablet
extract,
µg ml-1
Pure
DOX
added,
µg ml-1
Total
DOX
found,
µg ml-1
Pure DOX
recovered
(Percent±SD*)
DOX 100
2.97
2.97
2.97
1.5
3.0
4.5
4.44
5.98
7.37
98.24±1.09
100.3±1.26
97.84±1.42
0.49
0.49
0.49
0.25
0.50
0.75
0.74
1.01
1.26
100.1±1.76
104.7±2.36
102.6±1.85
1.96
1.96
1.96
1.0
2.0
3.0
2.93
3.88
4.95
97.38±2.15
95.87±1.95
99.62±2.38
DOXY 100
3.08
3.08
3.08
1.5
3.0
4.5
4.60
6.17
7.84
101.5±1.26
103.1±1.38
105.7±1.44
0.50
0.50
0.50
0.25
0.50
0.75
0.75
1.03
1.28
100.1±2.15
106.3±1.92
103.7±2.26
2.04
2.04
2.04
1.0
2.0
3.0
3.05
4.09
5.13
100.5±1.78
102.3±2.08
103.1±1.56 *Mean value of three determinations
107
SECTION 3.4
SIMPLE UV-VISIBLE SPECTROPHOTOMETRIC METHODS FOR THE
DETERMINATION OF DOXYCYCLINE HYCLATE IN PHARMACEUTICALS
3.4.1 INTRODUCTION
Ultraviolet spectroscopy or ultraviolet spectrophotometry (UV) refers to
absorption spectroscopy or reflectance spectroscopy in the ultraviolet spectral region.
Molecules containing π-electrons or non-bonding electrons (n-electrons) can absorb the
energy in the form of ultraviolet light to excite these electrons to higher anti-bonding
molecular orbitals [131]. UV spectrophotometry [132-135], because of simplicity,
reproducibility, speed and minimum requirement of solvent/reagent system, is widely
used for the assay of the therapeutic compounds used as medications. To the best of
author’s knowledge, no UV- spectrophotometric method has ever been reported for the
determination of DOX.
The present study reports the development and validation of spectrophotometric
methods with better detection ranges of DOX in pure form and in its solid dosage forms.
One UV spectrophotometric method in which the absorbance of the DOX in solution in
0.1 M HCl was measured at 240 nm and two visible spectrophotometric methods based
on the formation of either a greenish –yellow chromogen in 0.1 M NaOH peaking at 375
nm or yellow 2:1 complex formed by DOX with iron(III) in H2SO4 medium with an
absorption maxima of 420 nm were developed and successfully applied to the
determination of DOX in pure drug and in tablets. The developed methods were found to
possess several advantages in terms of sensitivity, selectivity, speed and cost-
effectiveness compared to the reported spectrophotometric methods. The details of the
methods are presented in this section 3.4.
108
3.4.2.0 EXPERIMENTAL
3.4.2.1 Apparatus
Shimadzu Pharmaspec 1700 UV/Visible and Systronics model 106 digital
spectrophotometers with 1 cm path length quartz cells were used for absorbance
measurements.
3.4.2.2 Reagents
All chemicals and reagents used were of analytical-reagent grade. Distilled water
was used throughout the investigation.
Hydrochloric acid (0.1 M): Prepared by successive dilutions of appropriate volume of
concentrated acid (S.D. Fine Chem, Mumbai, India, sp. gr. 1.18) in water.
Sodium hydroxide (0.1 M): One g of pure NaOH (S.D. Fine Chem, Mumbai, India) was
dissolved in water and diluted to 250 ml.
Sulphuric acid (0.05 and 0.01 M): Concentrated acid (S.D. Fine Chem, Mumbai, India,
sp. gr. 1.84) was diluted appropriately with water to get 0.05 and 0.01 M.
Iron(III) solution: A 0.5 % iron(III) alum solution was prepared by dissolving 1.25g of
pure ammonium iron(III) sulphate (S.D. Fine Chem, Mumbai, India) in 0.05 M H2SO4 in
a 250 ml calibrated flask.
Preparation of standard DOX solution
Standard drug solutions of 100 µg ml-1
in 0.1 M HCl for method A, 60 µg ml-1
in
0.1 M NaOH for method B and 200 µg ml-1
in 0.01 M H2SO4 for method C were
prepared by dissolving the calculated quantities of pure DOX in the specified solvents.
Tablets used are described in section 3.1.
3.4.3.0 ASSAY PROCEDURES
Method A
Varying aliquots (0.25, 0.5, 1.0, 2,0, 3.0, 4.0 and 5.0 ml of 100 µg ml-1
in 0.1 M
HCl) of standard solution corresponding to 2.5-50 µg ml-1
DOX were taken into a series
of 10 ml standard flasks, the content was diluted to the mark with 0.1 M HCl and mixed
well. The absorbance of each solution was then measured at 240 nm versus 0.1 M HCl.
Method B
Into a series of 10 ml calibration flasks, aliquots of DOX standard solution (60
µg ml-1
in 0.1 M NaOH) equivalent to 1.50-30.0 µg ml-1
DOX were accurately measured
109
and transferred and volume was made up to mark with 0.1 M NaOH. After mixing the
content, the absorbance of each solution was measured at 375 nm vs 0.1 M NaOH.
Method C
Different aliquots (0.0-5.0 ml) of DOX (200 µg ml-1
) were accurately measured
into a series of 10 ml calibrated flasks by means of microburette and the total volume was
adjusted to 5.0 ml with 0.01 M H2SO4. To each flask, 2 ml of 0.5% iron(III) alum
solution was added. The content was mixed and allowed to stand for 5 minutes and then
diluted to 10 ml with water. After mixing well, the absorbance was measured at 420 nm
against the reagent blank.
In all the cases, calibration curves were prepared and the concentration of the
unknown was read from the calibration graph or computed from the respective regression
equation derived using Beer’s law data.
3.4.3.2 Procedure for tablets
Method A
Twenty tablets were weighed and pulversized. A quantity of tablet powder
containing 10 mg of DOX was transferred into a 100 ml standard flask. The content was
shaken well with about 50 ml of 0.1 M HCl for 20 min. The mixture was diluted to the
mark with the same acid. It was filtered using Whatmann No 42 filter paper. First 10 ml
portion of the filtrate was discarded and suitable aliquots were subjected to analysis
following the procedure described earlier.
Method B
Tablet powder equivalent to 10 mg of DOX was transferred into a 100 ml
standard flask. The content was shaken well with about 50 ml of 0.1 M NaOH for 20 min
and diluted to the mark with 0.1 M NaOH. It was filtered using Whatmann No. 42 filter
paper. First 10 ml portion of the filtrate was discarded and subsequent portion was
analyzed after dilution to 60 µg ml-1
DOX with 0.1 M NaOH.
Method C
An accurately weighed portion of the tablet powder, equivalent to 20 mg of the
drug was shaken with 0.01 M H2SO4 in a 100 ml standard flask for 20 min. The mixture
was diluted to the mark with 0.01 M H2SO4, mixed well and then filtered through a
Whatmann No.42 filter paper. First 10 ml portion of the filtrate was discarded and a
110
convenient aliquot of subsequent portion was analyzed by the general procedure
described for pure drug.
3.4.3.3 Placebo blank synthetic mixture analyses
Twenty mg of the placebo blank prepared as described on section 3.1 was taken
and its solution was prepared as described under section 3.4.3.2, and then subjected to
analysis.
To 20 mg placebo blank of the composition described above, 20 mg of DOX was
added and homogenized, and the solution prepared as described under Section 3.4.3.2. A
convenient aliquot was then subjected to analysis by the procedures described above after
appropriate dilution.
3.4.4.0 RESULTS AND DISCUSSION
Spectral characteristics
DOX solution in 0.1 M HCl exhibited an absorption peak at 240 nm (Figure
3.4.1) and the absorbance at this wavelength was found to be linearly dependent upon the
concentration of drug whereas the drug solution in 0.1 M NaOH displayed a greenish
yellow colour peaking at 375 nm (Figure 3.4.1) serving as the basis for the quantification
of DOX. DOX was also found to react with iron(III) in H2SO4 medium yielding a stable
complex which was monitored at 420 nm (Figure 3.4.2). In all the cases, the
corresponding blank solutions showed negligible absorbance. Therefore these
wavelengths were used as analytical wavelengths throughout the investigation.
A
B
C
D
Figure 3.4.1 Absorption spectra of: A. DOX in 0.1 M HCl (24 µg ml
-1); B. 0.1 M HCl;
C. DOX in 0.1 M NaOH (11 µg ml-1
) and D. 0.1 M NaOH.
111
E
F
Figure 3.4.2 Absorption spectra of: E. DOX-iron(III) complex (30 µg ml
-1 DOX) and F.
iron(III) in sulphuric acid.
3.4.4.1 Method optimization
A series of preliminary experiments necessary for rapid and quantitative
formation of colored products to achieve the maximum stability and sensitivity were
performed in method B and method C. Optimum condition was fixed by varying one
parameter at a time while keeping other parameters constant and observing its effect on
the absorbance either at 375 nm in method B or at 420 nm in method C.
Method B.
Effect of NaOH concentration
The effect of NaOH concentration on the colour formation was investigated by
adding varying volumes of 0.1 M NaOH (0-5 ml) to a fixed amount of drug solution in
0.1 M NaOH. No difference in absorbance values was observed with increasing
concentration of NaOH in the range studied implying that additional NaOH had no role
on the color formation and its stability.
Effect of diluent solvent, standing time and the stability of the colored species
The effect of diluent was studied by using water and 0.1 M NaOH as diluting
solvents. NaOH showed good sensitivity compared to water and hence 0.1 M NaOH was
used as the diluent. Yellow color formed immediately after dissolution of drug in 0.1 M
NaOH and the color was stable for 2 h thereafter at laboratory temperature (30±20 C).
Method C
Effect of iron(III) solution
The effect of iron(III) concentration on the formation of DOX-iron(III) complex
was investigated by varying the volume of iron(III) solution, and using a fixed amount of
drug. The results revealed that the complex formation was unaffected in the range of
112
1-5.0 ml of 0.5% iron(III) solution in a total volume of 10 ml. Hence, 2 ml of 0.5%
iron(III) solution was used throughout the investigation.
Effect of sulphuric acid concentration on DOX- iron(III) complex formation
The effect of H2SO4 concentration on the complex formation was studied by
adding various amounts of 2 M sulphuric acid (0 - 4 ml) to a fixed amount of the drug
solution before mixing with iron(III) solution. The results revealed that complex
formation, sensitivity and stability were unaffected in the concentration range studied.
Reaction time and stability of the complex
The effect of reaction time after adding iron(III) solution and diluting to the mark
with water was studied. The color formation was complete in 5 min and stable upto 60
min thereafter.
Composition of DOX –iron(III) complex
The composition of the iron (III)-DOX complex was studied using Job’s
continuous variations method [136]. Drug and iron(III) solutions of 3.9 x 10-4
M each,
were prepared in 0.01 M H2SO4 and 0.05 M H2SO4, respectively, and mixed in various
molar ratios (with a total volume of 5 ml). The solutions were made upto mark, mixed
well and the absorbance was subsequently measured at 420 nm. The graph of the results
obtained (Figure 3.4.3) gave a maximum at a molar ratio of Xmax= 0.666 which indicated
the formation of a 2:1 (DOX:iron(III)) complex. The experiment was repeated three times
(n = 3) and in each case the formation constant (Kf) of the complex was calculated from
the continuous variation data using the following equation [137]:
[ ] n
M
n
m
m
fnCAA
AAK
)(/1
/2+
−=
where A and Am are the observed maximum absorbance and the absorbance value when
all the drug present is associated, respectively. CM is the molar concentration of drug at
the maximum absorbance and n is the stoichiometry with which iron(III) complexes with
drug. The average log Kf value (n=3) was found to be 7.08 with the percentage relative
standard deviation (%RSD) value of 0.75.
113
A
Am
Figure 3.4.3 Job’s continuous variations plot for iron(III)-DOX complex measured at
420 nm.
3.4.4.2 Method validation
Linearity and sensitivity
A linear correlation was found between absorbance at λmax and concentration of DOX in
the ranges given in Table 3.4.1. 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 3.4.1. The optical
characteristics such as Beer’s law limits, molar absorptivity and Sandell sensitivity values
of all the three methods are also given in Table 3.4.1. The limits of detection (LOD) and
quantification (LOQ) calculated according to ICH guidelines [47].
.
Table 3.4.1 Sensitivity and regression parameters
Parameter Method A Method B Method C
λmax, nm 240 375 420
Linear range, µg ml-1
2.5-50 1.5-30 10-100
Molar absorptivity(ε), l mol-1
cm-1
1.03 x 104 1.73 x 10
4 5.21 x 10
3
Sandell sensitivity*, µg cm
-2 0.05 0.03 0.098
Limit of detection (LOD), µg ml-1
0.28 0.17 1.28
Limit of quantification (LOQ), µg ml-1
0.84 0.52 3.87
Regression equation, Y**
Intercept (a) -0.0008 0.0036 -0.0023
Slope (b) 0.02 0.03 0.01
Standard deviation of a (Sa) 0.0998 0.1000 0.0025
Standard deviation of b (Sb) 0.0203 0.0009 4 x 10-4
Variance (Sa2) 0.01 0.01 6.25 x 10
-6
Regression coefficient (r) 0.9999 0.9999 0.9999 *Limit of determination as the weight in µg per ml 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.
114
Accuracy and precision
The assays described under “general procedures” were repeated 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) of the
methods. These assays were performed for three levels of analyte. The results of this
study are summarized in Table 3.4.2. The percentage relative standard deviation (%RSD)
values were ≤ 2.56% (intra-day) and ≤ 3.2% (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 DOX. Bias {bias % =
[(Concentration found - known concentration) x 100 / known concentration]} was
calculated at each concentration and these results are also presented in Table 3.4.2.
Percent relative error (%RE) values of ≤ 3.43% demonstrate the high accuracy of the
proposed methods.
Selectivity
A systematic study was performed to determine the effect of matrix by analyzing
the placebo blank and synthetic mixture containing DOX. The absorbance of the placebo
solution in each case was almost equal to the absorbance of the blank which revealed no
Table 3.4.2 Results of intra-day and inter-day accuracy and precision study
Method
DOX
taken,
µµµµg ml-1
Intra-day accuracy and
precision
(n=7)
Inter-day accuracy and precision
(n=5)
DOX
found ± CL,
µµµµg ml-1
%RE %RSD DOX found ±
CL, µµµµg ml-1
%RE %RSD
A
10.0
25.0
45.0
9.89±0.23
25.36±0.37
44.08±0.43
1.10
1.44
2.04
2.56
1.56
1.06
10.12±0.38
25.63±1.02
45.69±1.07
1.20
2.52
1.53
2.99
3.20
1.89
B
5.0
15.0
25.0
5.10±0.07
14.93±0.28
24.86±0.49
2.00
0.50
0.56
1.44
2.06
2.12
5.08±0.16
15.23±0.42
25.60±0.75
1.60
1.53
2.40
2.56
2.22
2.36
C
30.0
60.0
90.0
30.68±0.44
61.26±1.20
91.03±1.59
2.27
2.10
1.14
1.56
2.12
1.89
31.03±1.03
60.86±2.25
92.56±1.45
3.43
1.43
2.84
2.69
2.98
1.50 %RE. Percent relative error, %RSD. relative standard deviation and CL. Confidence limits were calculated from: CL = ± tS/√n. (The tabulated value of t is 2.45 and 2.77 for six and four degrees of
freedom respectively, at the 95% confidence level; S = standard deviation and n = number of
measurements).
115
interference. To assess the role of the inactive ingredients on the assay of DOX, a
synthetic mixture was analyzed. The absorbance resulting from the analysis of synthetic
mixture containing 25, 15 and 50 µg ml-1
DOX solution in method A, method B and
method C, respectively, were nearly the same as those obtained for pure DOX solutions
of identical concentrations. This unequivocally demonstrated the non-interference of the
inactive ingredients in the assay of DOX. Further, the slopes of the calibration plots
prepared from the synthetic mixture solutions were about the same as those prepared
from pure drug solutions.
Robustness and ruggedness
The robustness of the method (method C) was evaluated by making small
incremental changes in the volume of iron(III), and the effect of the changes was studied
by calculating the mean RSD values. The changes had negligible influence on the results
as revealed by small intermediate precision values expressed as % RSD (≤ 2.56%).
Method ruggedness was expressed as the RSD of the same procedure applied by four
different analysts as well as using four different instruments. The inter-analysts RSD
were within 3.0% whereas the inter-instruments RSD for the same DOX amount was less
than about 4.0% suggesting that the developed methods were rugged. The results are
shown in Table 3.4.3.
Table 3.4.3 Results of robustness and ruggedness expressed as intermediate precision ,
% RSD
Method DOX
taken*
Robustness Ruggedness
Parameter altered Inter-analysts,
(%RSD)
(n=4)
Inter-instruments,
(%RSD)
(n=4)
Volume of
iron(III)*
(%RSD)
A 25 - 2.89 2.45
B 20 - 2.55 2.98
C 60 2.56 3.00 3.35 *Volumes of iron (III) solution used were 1.8, 2.0 and 2.2 ml.
Analysis of tablets
The described procedures were successfully applied to the determination of DOX
in its tablets. The results obtained were statistically compared with the official BP method
[1]. The results obtained by the proposed methods agreed well with those of reference
method and with the label claim. The results were also compared statistically by a
116
Student’s t-test for accuracy and by a variance F-test for precision with those of the
reference method at 95 % confidence level as summarized in Table 3.4.4. 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.
*Average of five determinations.
Tabulated t value at the 95% confidence level is 2.77. Tabulated F value at the 95% confidence level is
6.39.
Recovery study
The recovery test was done by spiking the pre-analysed tablet powder with pure
DOX at three different levels (50, 100 and 150 % of the content present in the tablet
powder (taken) and the total was found by the proposed methods. Each test was repeated
three times. In all the cases, the recovery percentage values ranged between 96.45 and
103.4% with relative standard deviation in the range 0.27-1.83%. Closeness of the results
to 100 % showed the fairly good accuracy of the methods. The results are shown in Table
3.4.5.
Table 3.4.4 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
Proposed methods
Method A Method B Method C
DOX-T 100
100
101.3±0.73
100.8±0.48
t = 1.31
F = 2.31
101.6±0.36
t = 0.87
F = 4.11
100.4±1.42
t = 1.32
F = 3.40
DOXY 100
100
98.66±0.66
99.34±0.52
t = 1.82
F = 1.61
97.96±0.28
t = 2.35
F = 5.56
98.31±1.32
t = 0.56
F = 4.0
117
Table 3.4.5 Results of recovery study using standard addition method
Method A Method B Method C
Tablet
studied
DOX in
tablet
extract,
µµµµg ml-1
Pure
DOX
added,
µµµµg ml-1
Total
DOX
found,
µµµµg ml-
1
Pure DOX
recovered
(Percent±SD*)
DOX
in
tablet
extract,
µµµµg ml-1
Pure
DOX
added,
µµµµg ml-1
Total
DOX
found,
µµµµg ml-1
Pure DOX
recovered
(Percent±SD*)
DOX in
tablet
extract,
µg ml-1
Pure
DOX
added,
µg ml-1
Total
DOX
found,
µg ml-1
Pure DOX
recovered
(Percent±SD*)
DOX 100
20.16 20.16
20.16
10.0 20.0
30.0
30.47 40.48
50.85
103.1±0.34 101.6±0.52
102.3±0.48
10.16 10.16
10.16
5.0 10.0
15.0
15.20 20.31
25.28
100.7±0.62 101.5±0.44
100.8±0.51
40.16 40.16
40.16
20.0 40.0
60.0
60.46 80.44
102.20
101.5±1.62 100.7±0.85
103.4±1.36
DOXY
100
19.87
19.87
19.87
10.0
20.0
30.0
29.91
39.79
20.26
100.4±0.63
99.58±0.27
101.3±0.47
9.80
9.80
9.80
5.0
10.0
15.0
14.67
19.47
24.60
97.33±0.75
96.72±0.56
98.63±0.65
39.32
39.32
39.32
20.0
40.0
60.0
58.61
78.37
98.21
96.45±1.08
97.62±1.45
98.15±1.83 *Mean value of three determinations.
118
SECTION 3.5
DEVELOPMENT AND VALIDATION OF STABILITY INDICATING RP-HPLC
METHOD FOR THE DETERMINATION OF DOXYCYCLINE HYCLATE IN
PHARMACEUTICALS AND SPIKED HUMAN URINE
3.5.1 INTRODUCTION
HPLC is a chromatographic technique used to separate a mixture of compounds
in analytical chemistry and biochemistry with the purpose of identifying, quantifying and
purifying the individual components of the mixture. HPLC offers several advantages over
other techniques, including minimal sample manipulation before chromatography, rapid
analysis and the simultaneous analysis of multi-component mixtures with good
specificity, precision and accuracy. From the literature survey presented in Section
3.0.2.3 it is clear that there are only six reports on the HPLC determination of DOX in
pharmaceuticals. The reported methods for the determination of DOX in pharmaceuticals
have one or the other disadvantage (Table 3.6.1). No stability-indicating analytical
method has been reported for the determination of DOX in the presence of its degradation
products. In this section, the development and validation of an accurate, sensitive,
precise, rapid, isocratic reversed phase HPLC (RP-HPLC) method for the determination
of doxycycline hyclate in bulk drug and in tablets and also in spiked human urine has
been described. The best separation was achieved on a 250 mm x 4.0 mm i.d, 5.0 µm
particle size C8 reversed phase thermo column with acetonitrile-potassium
dihydrogenorthophosphate buffer (pH 4.0), 40:60 (v/v) as mobile phase. The details of
the method are presented in this section 3.5.
3.5.2.0 EXPERIMENTAL
3.5.2.1 Apparatus
HPLC analysis was performed on an Alliance Waters HPLC system equipped
with Alliances 2657 series low pressure quaternary pump, a programmable variable
wavelength UV-visible detector, Waters 2996 photodiode array detector and auto
sampler. Data were collected and processed using Waters Empower 2.0 software.
119
3.5.2.2 Chromatographic conditions
Chromatographic assay was performed using an C8 (5 µm, 4.0 × 250 mm i.d.,)
column. The mobile phase was composed of potassium acetonitrile-potassium
dihydrogenorthophosphate buffer (pH 4.0), 40:60 (v/v). The column effluent was
monitored on UV detector set at 325 nm.
3.5.2.3 Reagents
HPLC grade acetonitrile (Labscan Asia Co. Ltd, Bangkok, Thailand), analytical
reagent grade potassium dihydrogenorthophosphate (Rankem, Bangalore, India) and
potassium hydroxide (Rankem, Bangalore, India) were used. Deionised, Milli Q water
(Millipore, Bangalore, India) was used to prepare the mobile phase and diluent solutions.
Mobile phase: Mobile phase was prepared by mixing 0.01 M potassium
dihyrogenorthophosphate buffer adjusted to pH 4.0 with 0.1 M potassium hydroxide and
acetonitrile in the ratio 60:40 (v/v), and filtered through 0.4 micron membrane filter.
Diluent was a 1:1 mixture of potassium dihydrogenorthophosphate buffer and
acetonitrile.
Preparation of standard solution of DOX
A stock standard solution equivalent to 1000 µg ml-1
DOX was prepared by
dissolving accurately weighed amount of pure drug in the diluent solution.
Tablets used were the same described in section 3.1.
3.5.3.0 ASSAY PROCEDURES
3.5.3.1 Calibration graph
Ten µL of working standard solutions (30-300 µg ml-1
DOX) were injected
automatically onto the column in triplicate and the chromatograms were recorded. The
concentration of the unknown was computed from the regression equation derived using
the mean peak area and concentration data.
3.5.3.2 Urine sample
Five mg of pure DOX was added to 1 ml of drug-free urine, followed by 3 ml of
acetonitrile and the solution was allowed to stand for 2 min. Five ml of mobile phase was
added and then the solution was diluted to 10 ml with water. The content was allowed to
stand for 5 min and then centrifuged for 15 min at 4000 rpm. Finally, the solution was
filtered through 0.2 µm cellulose acetate syringe filter and the filtrate was made upto 25
120
ml with mobile phase. The resulting 200 µg ml-1
DOX and 100 µg/ ml (on dilution with
same solvent) solutions were subjected to analysis.
3.5.3.3 Procedure for tablets
Twenty tablets were accurately weighed and crushed into a fine powder and
mixed using a mortar and pestle. A quantity of tablet powder equivalent to 100 mg of
DOX was weighed accurately into a 100 ml calibrated flask, 50 ml of diluent solution
was added and was sonicated for 20 min to complete dissolution of the DOX, and the
mixture was then diluted to the mark with the diluent and mixed well. A small portion of
the resulting mixture (say 10 ml) was withdrawn and filtered through a 0.2 µm filter to
ensure the absence of particulate matter. The filtrate was appropriately diluted with the
diluent before injection onto the column.
3.5.3.4 Forced degradation procedure
Two portions of pure DOX each weighing 5 mg were separately spread uniformly
on two Petri dishes. One portion was kept in an oven at 105 0C and the other exposed to
the UV radiation of 360 nm, for 48 hours each. At the end of the stipulated time period,
the powders were transferred to two separate 25 ml calibrated flasks, dissolved in and
diluted to the mark with the diluent before the analysis. Into three separate 25 ml
calibrated flasks, 5 mg of pure DOX was weighed and 5 ml of 1 M HCl, 1 M NaOH or
10% H2O2 was added to the flasks. All three flasks were kept in hot water bath at 80 0C
for 3 hours. After cooling, the volume in each flask was made upto the mark with diluent
before subjecting to analysis.
3.5.4.0 RESULTS AND DISCUSSION
Mobile phase containing only acetonitrile or methanol was tried without success.
Sodium acetate and potassium dihydrogenorthophosphate in combination with either
acetonitrile or methanol in different volume ratios as organic modifiers were also tried.
The mobile phase consisting of 0.01 M potassium dihyrdogenorthophosphate with pH
adjusted to 4.0 using potassium hydroxide and acetonitrile (60: 40, v/v) was found ideal.
The selected mobile phase produced a well defined and resolved peak almost free from
tailing (tailing factor 1.2). The analysis was carried out at ambient temperature, which
besides being economical, offers many advantages like low column back pressure, good
chromatographic peak shape, improved column efficiency, higher theoretical plates and
121
consistency in retention time. Under the stated chromatographic conditions, the mean
retention time was 3.110 min (n=10). A model chromatogram is shown in Figure 3.5.1.
Further, the optimized chromatographic conditions were used to study the effect of forced
degradation of DOX after subjecting to various experimental conditions. Upon treatment
with 1 M NaOH, 1 M HCl or 1% H2O2, for 3 hrs at 80 0C, separately, there was no
change in the retention time and mean peak area in 1 M HCl whereas considerable
deviation from the above parameters was observed in 1 M NaOH (Figure 3.5.2) and 10%
H2O2 (Figure 3.5.3). There was no effect upon exposure to UV light at 1200 K flux and
thermal treatment at 105 0C, both for 48 hrs. All the three chromatograms of DOX after
acid, light and heat induced degradation were exactly similar to the typical chromatogram
of pure DOX (Figure 3.5.1).
Figure 3.5.1 Typical Chromatogram (Pure DOX, 300 µg ml-1
)
Figure 3.5.2 Chromatogram after treatment with 1 M NaOH (Pure DOX, 200 µg ml-1
)
122
Figure 3.5.3 Chromatogram after treatment with 10% H2O2 (Pure DOX, 200 µg ml-1
)
3.5.4.1 Method validation
Linearity and sensitivity
Working standard solution of DOX (1000 µg ml-1
) was appropriately diluted with
the diluent solution to obtain solutions in the concentration range 30-300 µg ml-1
DOX.
Ten µL of each solution was injected in triplicate onto the column under the operating
chromatographic conditions described above. The least squares method was used to
calculate the slope, intercept and the correlation coefficient (r) of the regression line. The
relation between mean peak area Y (n=3) and concentration, X expressed by the equation
Y = -10691.69 + 9811.60X, was linear. A plot of log peak area Vs log concentration was
a straight line with the slope of 0.9974 indicating excellent linearity between mean peak
area and concentration in the range 30 – 300 µg ml-1
DOX as shown in Table 3.5.1.
Sensitivity parameters such as limit of detection (LOD) and quantification (LOQ) were
estimated from the signal-to-noise ratio. The LOD defined as the lowest concentration
that gave a peak area with signal-to-noise ratio greater than 3:1, was found to be 0.02 µg
ml-1
. The lowest concentration that provided a peak-area with a signal-to-noise ratio 9.78,
which is called LOQ, was found to be 0.1 µg ml-1
.
Accuracy and precision
The method accuracy, expressed as relative error (%) was determined by
calculating the percent deviation found between concentrations of DOX injected and
concentrations found from the peak area. This study was performed by taking the same
123
three concentrations of DOX used for precision estimation. The intra-day and inter-day
accuracy (expressed as % RE) was better than 3.2% and the values are compiled in Table
3.5.2.
Method precision was evaluated from the results of seven independent
determinations of DOX at three different concentrations, 50, 100 and 150 µg ml-1
on the
same day. The inter-day and intra-day relative standard deviation (RSD) values for peak
area and retention time for the selected concentration of DOX were less than 1.3 and 0.5
%, respectively (Table 3.5.2).
Table 3.5.2 Results of intra-day and inter-day accuracy and precision study
DOX
injected,
µg ml-1
Intra-day accuracy and precision Inter-day accuracy and precision
DOX found
*, % RE % RSD
**, % RSD
*** DOX found
*, % RE %RSD
** % RSD
***
µg ml-1
µg ml-1
50.0 51.0 2.0 0.75 0.05 51.3 2.6 1.04 0.26
100.0 102.5 2.5 0.30 0.49 103.2 3.2 0.96 0.32
300.0 303.0 1.0 0.52 0.16 307.2 2.4 1.24 0.46
*Mean value of seven determinations.**Based on peak area.***Based on retention time
Robustness
To determine the robustness of the method small deliberate changes in the
chromatographic conditions like detection wavelength and column temperature were
made, and the results were compared with those of the optimized chromatographic
Table 3.5.1 Sensitivity and regression parameters
Parameters Value
Linearity range, µg ml-1
30-300
Slope (b) 9811.60
Intercept (a) -10691.69
Standard deviation of intercept (Sa) ± 36996.04
Standard deviation of Slope (Sb) ± 148.04
Correlation co-efficient (r) 0.9994
Limit of detection (LOD, µg ml-1) 0.02
Limit of quantification (LOQ, µg ml-1
) 0.10
Variance (Sa2) 6996.04
ntSa /±
41836.88
ntSb /±
168.33
ntSa /± =confidence limit for intercept, ntSb /± =confidence limit for slope.
124
conditions. The results indicated that changing the detection wavelength (±1 nm) had
some effect on the chromatographic behavior of DOX. However, the alteration in the
column temperature (±1 0C) had no significant effect. The results of this study expressed
as % RSD are summarized in Table 3.5.3.
Selectivity
Method selectivity was checked by comparing the chromatograms obtained for
pure DOX solution, synthetic mixture, tablet solution and placebo blank. An examination
of the chromatograms of the above solutions revealed the absence of peaks due to
additives present in tablet preparations.
System suitability
System suitability parameters were measured to verify the system performance
and the values of retention time, number of theoretical plates and tailing factor were
3.113 ± 0.0049, 5180 per column and 1.2, respectively. All the values were within the
acceptable range.
Solution stability
The stability of standard and sample solutions was determined by monitoring the
peak area and retention time over a period of 24 hrs by injecting the solutions every 8 h.
The standard and sample solutions were stored at ambient temperature (25 0C) and
protected from light during the stability study. No changes in drug concentrations were
observed over a period of 24 hrs as shown by the small % RSD values. The % RSDs for
peak area (n = 4) was 1.4% for pure drug solution and the value for retention time (n= 4)
was 0.14%. The results are presented in Table 3.5.4. No significant changes in
concentration of the active ingredient were observed in the tablet solution as well
125
Table 3.5.3 Results of robustness study (DOX concentration, 200 µg ml-1, n=3) expressed as intermediate precision (%RSD)
Chromatographic
condition
Modification
Peak area precision (n=3)
Retention time precision (n=3)
Area
Mean area ±
SD
Standard
error of
mean
% RSD
Retention
time,
min
Mean RT ±
SD,
(min)
Standard
error,
min
%RSD
Wavelength (nm) 324 2255108 2.823
325 2200112 2202238.3 ±
173866.8 29929.39 0.0231 2.819
2.818 ±
0.005 0.0029 0.0018
326 2151495 2.813
Column temperature (
0C)
26 2373643 2.822
25 2371524 2371667.3 ±
1907.89 1101.61 0.0008 2.823
2.823 ± 0.001
0.0006 0.0004
24 2369835 2.824
Table 3.5.4 Solution stability (DOX concentration was 200 µg ml
-1)
Time, h Peak area Retention time (min)
0 2138277 3.11
8 2145671 3.12 16 2246830 3.10
24 2378480 3.09
126
Application to urine sample and tablets
The developed and validated method was successfully applied to determine DOX
in spiked urine sample with satisfactory recovery (Table 3.5.5). The results obtained
tallied closely with the labeled amount in the case of tablets (Table 3.5.6), thus indicating
the utility of the method for content uniformity evaluation. The results were statistically
compared with those obtained by official method [1] for accuracy and precision by
applying the Student’s t-test and variance ratio F-test. The calculated t- and F- values
were less than the tabulated values of 2.77 and 6.39 at the 95% confidence level and for
four degrees of freedom suggesting that there was no significant difference between the
proposed method and the reference method with respect to accuracy and precision.
*Mean for five determinations.
*Mean value of five determinations
** Marketed by: 1. MicroLabs Pvt. Ltd., Bangalore, India, 2. Dr. Reddy’s Laboratory, Bangalore, India.
Figure in the parenthesis are the tabulated values for four degree of freedom at 95% confidence level.
Recovery studies
To further assess the accuracy and reliability of the method, recovery studies via
standard addition method was performed. To the pre-analyzed tablet powder, pure DOX
was added at three levels and the total was found by the proposed method. Each test was
triplicated. When the test was performed on 100 mg tablets, the percent recovery of pure
DOX was 106.0 with standard deviation of 0.12. The results indicated that the method is
Table 3.5.5 Results of DOX recovery studies in spiked urine sample
Spiked concentration
(µg ml-1
)
Found ± S.D* % Recovery ± RSD
*
100.0 101.4 ± 0.005 101.4 ± 0.005
200.0 206.4 ± 0.012 103.2 ± 0.033
Table 3.5.6 Results of determination of DOX in tablets and statistical comparison with the reference method
Tablet
brand
name**
Nominal
amount,
mg
Found* (Percent of label claim ± SD)
Reference
method
Proposed
method
Student’s t-
value (2.77)
F-value
(6.39)
Doxy
1
Doxt
2
100
104.8 ± 0.67 106.0 ± 1.22 2.00 3.31
100 98.46 ± 0.83 99.38 ± 1.42 1.29 2.93
127
very accurate and that common excipients found in tablet preparations did not interfere.
The results are complied in Table 3.5.7.
Table 3.5.7 Results of recovery study by standard addition method
Tablet
DOX in
tablet, µg
ml-1
Pure DOX
added,
µg ml-1
Total found,
µg ml-1
Pure DOX recovered*,
Percent ± SD
Doxy 100 mg
Doxt
100 mg
106.0 50 159.95 107.90 ± 0.56
106.0 100 212.95 106.95 ± 0.72
106.0 150 265.98 106.65 ± 0.86
99.38 50 150.03 101.30 ± 0.64
99.38 100 201.88 102.50 ± 0.46
99.38 150 250.28 100.60 ± 0.92
*Mean value of three determination
128
SECTION 3.6
CONCLUSIONS ON CHAPTER III-Assessment of the Methods
A comparison of performance characteristics of the proposed titrimetric, spectrophotometric and HPLC methods with those of the
existing methods is presented in Table 3.6.1.
Table 3.6.1 Comparison of performance characteristics of proposed methods with the existing methods
A. Titrimetry
B. Spectrophotometry
Sl.
No. Reagent/s used Methodology
λλλλmax
(nm)
Linear range
(µg ml-1
)
l mol-1
cm-1
LOQ
(µg ml-1
) Remarks Ref.
1 a) Copper carbonate Complex colour measured 395 10.0-80.0
mg ml-1 -
FIA assembly required and
least sensitive 4
b) Chloramine-T Oxidation of drug in alkaline
medium and red coloured product
measured
525 5.37 x 10
-5 to
7.16 x 10-4 -
FIA assembly required and
least sensitive
c) 4-Aminophenazone and potassium
hexacyanoferrate(III)
Colour of the dye measured 520 - - FIA assembly required and
the pH dependent
Sl. No. Reagent Titration conditions Range, mg Remarks Ref. No.
No titrimetric method has been reported for DOX
1 Perchloric
acid and
mercuric
acetate
a. Visual titration of DOX anhydrous acetic acid against
0.01 M perchloric acid using crystal violet as indicator
b. Potentiometric titration of DOX in anhydrous acetic
acid against 0.01 M perchloric acid
4.0-40 mg Applicable over
wide linear range
This
work
2 KBrO3-KBr
mixture
Iodometric back titration method in HCl medium 1.0-8.0 mg Uses an oxidant
which is highly
stable solution
This
work
129
2 Thorium(IV) Yellow complex measured 398 0.4-3.2
- pH dependent and narrow
linear range 5
3 Sodium cobaltnitrite and acetic acid
Colour forming reaction
243 0.01-0.03 mg ml
-1
-
Heating required. Less sensitive
6
4
Uranyl acetate-DMF medium
1:1 Complex formation reaction 405 0-135 - Requires organic solvent,
less sensitive 7
5 Cu(II)/H2O2-alkaline
medium Degradation study 510
2.97-17.78
1.89
Use of buffers, scrupulous
control of experimental variables and special
equipment for kinetic
measurement required
8
6 DMF/NaOAc-AcOH
buffer (pH 4.5)
Partial least squares multivariate
calibration method
277-
349
1.7-42
-
Require special equipment,
Use of organic solvent, pH dependant
9
7.
a) KBrO3-KBr
/HCl
and methyl orange
Bromination and oxidation of
drug and determination of
unreacted Br2 with methyl
orange
520 0.25-1.25
2.62 x 105
0.07 Highly sensitive, non-
stringent optimum
conditions used, simple
instrument employed.
This
work
b) KBrO3-KBr
/HCl
and indigo carmine
Bromination of drug and
determination of unreacted Br2
with indigo carmine
610 0.5-5.0
6.97 x 104
0.27
8. F-C reagent/Na2CO3 Blue colored chromogen was
measured 770
0.75-12
2.78 x 104
0.20/0.08 No heating or extraction.
More sensitive
This
work
9. a) 0.1 M HCl UV spectrophotometric
detection 240
2.5-50
1.03 x 104
0.84 No additional reagent.
Simple instrument
employed. This
work
b) 0.1 N NaOH Greenish-yellow coloured
product 375
1.5-30
1.73 x 104
0.52
c) Fe(III) ammonium
sulphate-acid medium Yellow complex measured 420
10-100
5.21 x 103 3.87
Mild acidic conditions
used; stable colour
measured and simple
instrument employed
130
C. HPLC Methods
Sl.
No. HPLC Conditions UV detection
Linear
range,
(µg ml-1
)
LOD
(µg ml-1
) RSD Remarks Reference
1 Lichrosorb RP-8 (250 mm’4.6 mm, 10 mm
particle size); methanol:acetonitrile: 0.01M oxalic
acid (2:3:5, v/v); flow rate: 1.25 ml/ min
350 25.20-252 1.15 >2% Three component mobile
phase, applied to veterinary
pharmaceutical samples
31
2 Hamilton RP-1 (25 x 0.46, cm, i.d.);
tetrahydrofuran:0.2M phosphate buffer (pH 8.0):0.2M tetrabutylammonium hydrogen sulphate
(pH 8.0): 0.1M sodium acetate (pH 8.0): water
(6:10:5:1:78), flow rate: 1.00
254 60 -- >2% Multi constituents of mobile
phase, elevated temperature
32
3 Porous graphitic carbon column; 0.05M potassium
phosphate buffer (pH 2.0):acetonitrile (40:60),
flow rate: 1.00
268 5.0-50 2.00 >2% Applied to the assay in a
multicomponent mixture
33
4 Chromolith flash RP-18e, 25-4.6 mm; acetronitrile:water (20:90, v/v) pH 2.5 adjusted
with 98% H3PO4, flow rate: 0.48
213 0.5-2.0 >2% Narrow range, sequential injection setup required
34
5 A polystyrene-divinylbenzene column and a polymethaxrylate column with octadecyl ligands.
Acetonitrile-0.02 M sodium perchlorate (pH 2.0)
-- -- -- -- -- 36
6 Hypersil BDS C8 (250 mm x 4.0 mm i.d, 5.0 µm
particle size) thermo column; potassium
dihydrogen orthophosphate buffer (pH-4.0)-
acetonitrile (60:40) (v/v), flow rate: 1.00
325 30.0-300 0.1 <1% Wide linear dynamic range,
low LOD, low RSD (%),
simple mobile phase and
first application to stability-
indicating assay
Present
method
131
A comparison of performance characteristics of the proposed methods with those of
the existing methods is presented in Table 3.6.1 above. To the best of the author’s
knowledge, no titrimetric method has ever been reported. Because of the scarcity of
titrimetric procedure, the author developed three simple, selective and rapid titrimetric
procedures for the determination of DOX in formulations. The first two reactions are based
on neutralization reaction and are specific for the amino group present in DOX. The
methods are applicable over a wide linear range of 4.0-40 mg DOX, although the methods are
somewhat less sensitive. It should be pointed out that the non-aqueous titrimetric
procedures cannot be applied directly to some of the tablet preparations since interference
from some excipients was encountered. However, this could be overcome by extracting the
drug with methanol and reconstituting the sample with acetic acid after evaporating
methanol. The proposed titrimetric method employing bromated-bromide mixture has the
advantages of simplicity, speed, accuracy and precision and the use of inexpensive
equipments. The method is sensitive and applicable over 1.0-8.0 mg DOX.
Spectrophotometry is found to be the most commonly used technique for the assay of
DOX. The author has developed six more spectrophotometric methods for the determination
of DOX in bulk and in pharmaceutical formulations. The outstanding performance
characteristics of the proposed methods are simplicity, sensitivity and wide dynamic linear
concentration range of applicability. Among the proposed methods, the method employing
KBrO3-KBr-methyl orange is the most sensitive method with ε value of 2.62 × 105
l mol-1 cm-1
and the method using Fe(III) ammonium sulphate in acid medium quantifies DOX concentration
over a widest linear dynamic range. Eventhough, the bromated-bromide solutions are highly
stable, the reactions are not specific and are not ideally suited for assay in combined dosage
forms, where any substance with active hydrogen and unsaturation would cause interference.
Fortunately, the dosage forms used in the present study were devoid of such species as
shown by the results of assay as well as of recovery experiments. The proposed methods are
much simpler than the existing spectrophotometric methods with respect to optimum
conditions. They do not involve stringent experimental conditions unlike the reported
methods [4-9]. They rely on the use of simple and inexpensive chemicals.
The author has developed a HPLC method which uses a simple mobile phase
compared to the multi-component mobile phase in many reported methods. The separation
132
and determination was achieved at an ambient temperature relative to elevated temperatures
in a couple of reported HPLC methods. This itself offers the advantages of low column back
pressure, good peak shape, improved column efficiency, higher theoretical plates and
consistent retention time. The sensitivity expressed in LOD is better than all the reported
HPLC methods and the RSD (%) of <1% attained in the method is far better than obtained in
other methods. Furthermore, this is the first stability-indicating method ever reported for
DOX. In addition to solution stability as a function of time, forced degradation under a
variety of stress-conditions has been studied thereby widening the application. The small
retention time (about 3 min) and runtime (5 min) enable rapid determination which is
important in routine analysis. Simple mobile phase and low flow rate (1 ml min-1
) make the
method attractive since these features help in saving cost and time of analysis.
Selectivity of all the proposed methods was examined by both placebo blank and synthetic
mixture analyses. The methods were found to be highly selective, since, there was no
interference from commonly employed tablet excipients. The selectivity of the HPLC method
is further shown by the applicability of the method in spiked human urine sample. With the
relative error (RE) and relative standard deviation (RSD) values of under 3.5%, all the
titrimetric and spectrophotometric methods are fairly accurate and precise. The proposed
titrimetric and spectrophotometric methods rely on the use of inexpensive and eco-friendly
chemicals, and simple instrumentation.
Thus, three titrimetric, six spectrophotometric and one HPLC methods for the assay of
DOX in pharmaceuticals have been developed and validated according to the current ICH
guidelines. The methods have been demonstrated to be fairly accurate and precise in addition to
being highly sensitive. The titrimetric and spectrophotometric methods can usefully be
employed in routine use in areas /countries which lack modern instrumental facilities such as
HPLC, LC-mass spectrometry, spectrofluorimetry, capillary electrophoresis, etc.,
133
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