CHAPTER 3 TITRIMETRIC, SPECTROPHOTOMETRIC …shodhganga.inflibnet.ac.in/bitstream/10603/36434/7/chapter 3.pdf · Doxycycline hyclate ... hydrogen sulphate (pH 8.0): 0.1M sodium acetate

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


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


3.2.2.1 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


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.


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.


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


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


DOX

taken

(µg ml-1

)


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



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


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.


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


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.


3.3.2.1 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.


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.


3.3.3.1 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.


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.


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


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


0.125-1.25 0.5-5.0


cm-1

2.62 ×105

6.97 × 104

Sandell sensitivity*, µg cm

-2 0.002 0.010


0.02 0.091


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.


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


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.



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


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)


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.



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


3.4.2.1 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.


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.


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.


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.


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.


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].

.


Parameter Method A Method B Method C

λmax, nm 240 375 420


2.5-50 1.5-30 10-100


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


0.28 0.17 1.28


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


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


Method

DOX

taken,

µµµµg ml-1


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.


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*


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)


Reference

method

Proposed methods


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


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.


3.5.2.1 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.


3.5.3.1 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.


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.


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

)


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

.


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).


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


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


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.


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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