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Chapter 4

Doxofylline

Page no. 36 to 63

Chapter 4 Doxofylline

36

Chapter 4

Doxofylline

Doxofylline, [7-(1, 3-dioxolan-2-ylmethyl)-3, 7-dihydro-1, 3-dimethyl-1H-purine-2, 6-

dione] is a new bronchodilator xanthine based drug which differs from theophylline by the

presence of dioxalane group at position 7. It is used in the treatment of bronchial asthma,

chronic obstructive pulmonary disease (COPD), and chronic bronchitis [49-51]. The

mechanism of action is similar to that of theophylline in that it inhibits phosphodiesterase

(PDE-IV), thereby preventing breakdown of cyclic adenosine monophosphate (cAMP).

Increase in cAMP inhibits activation of inflammatory cells resulting in bronchodilating

effect [52]. In contrast to theophylline, doxofylline has very low affinity towards adenosine

A1 and A2 receptors which explain its better safety profile [53, 54].

4.1 Literature review of analytical methods

Although various bioanalytical methods for estimation of doxofylline in human serum [55–

58], stability indicating HPTLC method [59], HPLC [60, 61] and HPLC method for

estimation of doxofylline in dosage form [62] are reported in the literature, there have

been no reports of characterization of the degradation products. Therefore, in this chapter

the stability properties of the drug doxofylline under different stress conditions were

studied. Structure of degradation products formed under hydrolytic conditions was

confirmed by IR, Mass and NMR spectra and mechanistic degradation pathways were

proposed.

4.2 Experimental work

4.2.1 Preparation of sample

4.2.1.1 Preparation of 0.1 N sodium hydroxide

0.4 g of sodium hydroxide (NaOH) was dissolved in water and made up to 100 mL to get

0.1 N sodium hydroxide solution.


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4.2.1.2 Preparation of 0.1 N hydrochloric acid

0.85 mL of concentrated hydrochloric acid (HCl) was diluted with water and volume

adjusted to 100 mL to get 0.1 N hydrochloric acid solution.

4.2.1.3 Preparation of 3% hydrogen peroxide

To 10 mL of hydrogen peroxide solution (30% w/v H2O2) water was added and volume

adjusted to 100 mL to get 3% hydrogen peroxide solution.

4.2.1.4 Preparation of 0.01 M potassium dihydrogen orthophosphate buffer (pH 3)

1.36 g of KH2PO4 was dissolved in 1000 mL of water and pH was adjusted to 3.0 with

ortho-phosphoric acid.

4.2.2 Forced degradation study

Stress studies were carried out as per ICH guidelines for stress conditions. A minimum of

four samples were generated for every stress condition for each drug, viz., the blank

solution stored under normal conditions, the blank subjected to stress condition in the same

manner as the drug, zero time sample containing the drug (which was stored under normal

conditions), and the drug solution subjected to stress treatment. A preliminary study was

performed to gain some basic information about the stability of the compound. Then in the

final study the drug was stressed to maximum condition where 5-20% degradation

occurred. The drug was declared as stable if no degradation occurred after 7 days of stress.

The same stress degradation study was performed on marketed tablet formulation of

doxofylline, Doxoril (Macleods Pharma) to determine formation of any degradation

product due to drug-excipient interaction.

The specific stress conditions were as follows:

4.2.2.1 Hydrolytic studies

Hydrolytic studies were carried out at acidic, neutral and basic conditions by heating it in

1N HCl at 500 C for 30 h, 0.1N NaOH at 500 C for 8 h, and in distilled water at 700 C for 7

days. The drug concentration was 1mg mL−1 for each study.


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4.2.2.2 Oxidative studies

For oxidative study drug was dissolved in 3% H2O2 at concentration of 1mg mL-1 and in

30% H2O2 at concentration of 1 mg mL-1 and kept at room temperature for 7 days.

4.2.2.3 Photolytic study

The drug was taken in petri-plate to make a thickness of 1mm and exposed at 400 C to 1.2

x 106 lux h of fluorescent light and 200 watt h m-2 of UV light [15]. Dark control was run

simultaneously.

4.2.2.4 Thermal stress

The drug was spread in Petri dish to make 1mm layer and was heated at 700 C for 15 days

in dry air oven.

4.2.3 HPLC Analysis

All the stressed samples were diluted with mobile phase to make concentration of 100g

mL-1 and injected on HPLC. Phenomenex Gemini C18 (250mm×4.6mm, 10m particle

size) column was used for analysis. Mobile phase containing 20 volumes of acetonitrile

and 80 volumes of potassium dihydrogen orthophosphate(0.01M) previously adjusted to

pH 3 with ortho-phosphoric acid was used. Analysis was carried out at 274nm. Flow rate

of mobile phase was kept at 1mL min-1 and injection volume was 20L.

Another gradient method was developed in order to check the possibility of any late

eluting degradation product. The mobile phase consist of gradient program of solvent A

and B. Solvent A composed of mixture of potassium dihydrogen orthophosphate (10mM)

with its pH adjusted to 3 with orthophosphoric acid and acetonitrile in ratio of 95:5 (v/v) .

Acetonitrile and water in the ratio of 95:5 (v/v) was used as solvent B. The flow rate of the

mobile phase was 1.0 ml/min with a linear gradient program of 0/20, 5/20, 7/30, 9/50,

16/75, 20/65, 24/55, 26/40, and 30/20 (time (min)/%B). Analysis was carried out at

274nm.

4.2.4 Semi-preparative HPLC

The analytical method developed was scaled to semi-preparative HPLC in order to isolate

the degradation products formed during stress study. As the main aim of isolation was to


39

characterize the degradants, more quantity of drug was taken for stress so as to generate the

quantity of degradants sufficient for characterization by different spectroscopic techniques.

One gram of accurately weighed doxofylline was dissolved in 1000 mL of 1N HCl and

kept for hydrolysis for 30hr at 500 C. For isolation of base degradation product 1gm of

doxofylline was added in 1000 mL of 0.1 N NaOH and kept for hydrolysis at 500 C for 8

hr. The mobile phase consisted of water and acetonitrile in the ratio of 80:20 (v/v) and pH

was adjusted to 3 with formic acid. The column used was Phenomenex Luna C18

(250mm×10mm, 10m particle size) and the flow rate was increased to 4.2mL min-1. The

injection volume was increased to 200L. The organic phase from the collected fractions

was evaporated by using Buchi rotavapor. The aqueous sample was then freezed at -800 C

and then kept in lyophilizer to remove water. The lyophilized sample was then dissolved in

dichloromethane and in that anhydrous sodium sulphate was added in order to remove any

traces of water. It was then filtered and dichloromethane evaporated using rotavapor. The

sample was again treated with hexane and ethyl acetate to remove any traces of impurity.

The final degradants obtained were used to characterization purpose.

4.2.5 Characterization of degradation products

4.2.5.1 Mass spectroscopy

The degradation products isolated by semi-preparative HPLC were dissolved in methanol

and injected by direct infusion (2L) through LC-MS (Perkin Elmer API-165) using

acetonitrile and 0.1 % ammonium acetate (90:10 v/v) as a mobile phase at 0.2 mL min-1

flow rate. The fragmentation pattern of the drug and degradation products was carried out

using API-2000LC/MS-MS triple quadrupole system with parameters: ionization source

(+ve ESI), nebulizer pressure (nitrogen 20 psi), gas temperature (2500C), drying gas

(nitrogen 30 psi) and capillary voltage (5500 V). The data was processed using Analyst 1.4

software.

4.2.5.2 FT IR Spectroscopy

The IR spectra of drug and degradation products were recorded using Shimadzu IR

Affinity 1 FT-IR instrument and IR solution software. Around 1mg of sample was

thoroughly mixed with 100 mg of finely powdered KBr. The diffuse reflectance spectra

were recorded using DRS assembly which is similar to transmittance spectra.


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4.2.5.3 NMR Spectroscopy

1H and 13C-NMR spectra of doxofylline and its degradation products were recorded by

using Bruker NMR 300MHz instrument with a dual broad band probe and z-axis gradients.

Spectra were recorded using DMSO-d6 as a solvent and tetramethylsilane as an internal

standard.

4.2.6 Validation of analytical method

4.2.6.1 Linearity and range

A stock solution of the drug was prepared at strength of 1mg mL−1. It was diluted to

solutions of concentration in the range of 5–30g mL−1 of the drug. The solutions were

injected in triplicate into the HPLC column, keeping the injection volume constant (20L)

4.2.6.2 Specificity

Specificity of the HPLC method was determined by spiking the degradation products at

0.15% level and determining the resolution. Peak purity of drug and its degradation

product was determined by PDA detector to rule out overlap of peaks.

4.2.6.3 Precision

Six injections of three different concentrations each (10, 20 and 30g mL−1) of bulk drug

were injected and analyzed on the same day and the values of relative standard deviation

(R.S.D.) were calculated to determine intra-day precision. These studies were also repeated

on different days to determine inter-day precision.

4.2.6.4 Accuracy

Accuracy was determined by spiking a mixture of stressed samples with three known

concentrations of the drug i.e., 10, 20 and 30 g mL-1 in triplicate and % recoveries of the

added drug were calculated.


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4.3 Result and Discussion

4.3.1 Stress degradation study

The chromatogram of pure doxofylline shows retention time of 7 min is as shown in Fig

4.1a. Two degradation product peaks were observed, one peak DP-I in acid (10.92%) and

another peak DP-II in basic conditions (7.62%) respectively (Fig. 4.1b, c). No degradation

products were generated on heating the drug in water for 7 days at 700 C (Fig.4.1 d),

indicating it to be stable under neutral condition. Negligible degradation occurred in

oxidative stress (Fig. 4.1e) and no degradation observed in thermal (Fig. 4.1f) and

photolytic condition (Fig. 4.1g). Stress study carried out on formulation under identical

conditions showed no peak due to drug-excipient interaction indicating there is no

incompatibility between drug and excipients.


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Fig. 4.1 Chromatograms of a) Doxofylline and its degradation under b) acidic, c) basic, d) neutral

e) Oxidative, f) thermal, g) photolytic and h) drug spiked with degradation products using isocratic

method


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The gradient method also shows the similar results and there was no late eluting impurity

in 30 minute runtime. One degradation product was observed in acid hydrolysis with

11.2% degradation and other in base hydrolysis with 8% degradation, while it was found

stable in other stress conditions (Fig 4.2).

Fig. 4.2 Chromatograms of a) Doxofylline and its degradation under b) acidic and c) basic

conditions using gradient programming

4.3.2 Isolation of degradation products

The isolated degradants from acid and base hydrolysis were analyzed by HPLC to check

the purity. But in both cases small peak of drug was seen along with degradants, hence

again purified by semi-preparative HPLC. The purity of degradation product I and II was

99.8 and 99.9 respectively.


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4.3.3 Characterization of degradation products

The structures of isolated pure degradation products were characterized by NMR, mass

spectra and functional groups were identified by IR spectra. The spectra of doxofylline

were compared to that of degradation products in order to ascertain the changes occurred in

drug due to degradation. Molecular structure of the drug and its degradation products has

been numbered to compare the NMR spectra of degradation products with the parent

molecule.

4.3.3.1. Spectral data of parent drug doxofylline

The ESI mass spectrum exhibited a protonated molecular ion peak at m/z 267 in positive

ion mode indicating the molecular weight of 266. The tandem mass spectrum showed the

fragment ions m/z 223, 181.2, 166.2, 138.1, 124.1 and 87.1(Fig 4.3).

The FT-IR spectrum, two strong peaks at 1697cm-1 and 1658cm-1 indicated presence of

two carbonyl groups. A strong peak at frequency 1546cm-1 indicated presence of C=N

stretch. A medium peak at 1232cm-1 was due to C-O stretch (Fig 4.4).

Fig. 4.3 Fragmentation pattern of doxofylline


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Fig. 4.4 FT-IR spectrum of doxofylline

The 1H NMR and 13C NMR spectra were recorded using DMSO-d6 as a solvent are as

shown in Fig.4.5. The spectral assignment for proton and carbon signals chemical shift

values has been shown in table 4.16 in detail.


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Fig. 4.5 1H-NMR and 13C-NMR spectrum of doxofylline


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4.3.3.2 Structure elucidation of the degradation product I (DP-I)

The MS analysis showed the molecular ion peak (M+H) of DP I at m/z 255 (Fig 4.6). The

chemistry guided approach suggests the possibility of breaking of dioxalane ring and

formation of acetaldehyde (Mol. wt. 222). In presence of moisture the aldehyde is hydrated

to give 1, 1 diol [Mol.Wt.240] which is a reversible reaction. [63] As the sample was

dissolved in methanol for mass, there was formation of pseudomolecular ion with

methanol (M+H+MeOH) [64] having molecular weight 254, indicated the presence of diol

group attached to the purine moiety (Scheme 4.1). Further confirmation of molecular

adduct formation was obtained by recording mass spectra in ethanol which showed

molecular ion peak (M+H+EtOH) at m/z 269 due to addition of ethyl group coming from

ethanol (Fig 4.7).

Fig 4.6 Mass spectra of DP-I (in methanol)


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Fig 4.7 Mass spectra of DP-I (in ethanol)

Scheme 4.1 Mechanism of molecular adduct formation in DP-I

Fragmentation pattern of DP-I showed major fragment ions m/z 223, 181 and 124 and it

resembles the probable fragmentation pattern of suggested structure (Fig 4.8).


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Fig 4.8 Fragmentation pattern(MS2) of DP-I

The IR spectrum of DP-I was taken and compared with spectra of doxofylline. A peak at

3365 cm-1 indicates the presence of hydroxyl group. Presence of two carbonyl peaks at

1712cm-1 and 1666 cm-1 signify the existence of purine ring similar to the parent drug as

shown in Fig 4.9.


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Fig 4.9 FT-IR Spectrum of DP-I

1H -NMR spectrum of DP-I showed loss of two protons as compared to parent compound.

There was disappearance of proton signals at 3.79 ppm (4’) and 3.82 ppm (5’) which

was attributed to the loss of ethylene group. A negligible shift in N-methyl proton signals

at 1, 3 positions and methine proton at position 8 indicated that the purine ring remained

intact. There were two extra proton signals found at 6.18 ppm (3’ and 6’ position) as

shown in Fig 4.10.


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Fig. 4.10 1H-NMR of DP-I

In 13C- NMR of DP-I, the disappearance of carbon signals at 64.5 ppm (4’ and 5’

position) of ethylene group indicated dioxalane ring opening. A negligible shift in the

carbon signals (C-2 to C-8) indicated that the purine ring remained intact. Thus the NMR

suggested the structure which fits well with the structure as shown in above Fig. 4.11 (m/z

240).


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Fig 4.11 13C-NMR of DP-I

4.3.3.3 Structure elucidation of the degradation product II (DP-II)

The ESI mass spectrum of DP II exhibited the protonated molecular ion peak at m/z 241 in

positive ion mode indicating the loss of 26 amu when compared with doxofylline which is

having molecular weight 266. As the pyrimidine ring of xanthine is more susceptible to

base hydrolysis, there is a possibility that the ring will break at either at C-2 or C-6

position. The two possible structures of degradation products are shown in Fig. 4.12.


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Fig 4.12 Mass spectra of DP-II

The major fragments ions m/z 210, 184, 138, 124 and 87 (Fig 4.13) fits well for the

probable fragmentation pattern of DP-II (scheme 4.2).

The IR spectra of DP- II a sharp peak at 3278cm-1 indicated the presence of amino group.

Absence of peak at 1697 cm-1 clearly indicated that only one carbonyl group is present.

The shift of another carbonyl group from 1658cm-1 in parent drug to 1612cm-1 in DP-II

was due to conjugation effect. The peak due to C-N stretching was observed at 1369 cm-1.

(Fig 4.14).


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Fig 4.13 Fragmentation pattern(MS2) of DP-II

Fig 4.14 FT-IR Spectra of DP-II


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1H NMR spectrum showed appearance of two extra proton signals, one at 7.08 and

another at 5.2 ppm. There was a negligible shifts in proton signals from 1’ to 6’ positions

indicates the dioxalane ring remained intact. A slight shift from 7.97 to 7.34 ppm is

observed in the proton signal at 8th position. The upfield shift of two methyl proton signals

from 3.37 to 2.75 ppm at 1st and 3.17 to 2.70 at 3rd position was seen. All these

observations suggested that there was purine ring opening (Fig 4.15).

Fig. 4.15 1H-NMR of DP-II

In 13C-NMR, there is negligible shift for carbons present in the dioxalane ring confirm

there was no change in dioxalane ring. The disappearance of carbon signal at 150 ppm

which was attributed to carbonyl functionality (2nd position) confirmed the opening of the

purine ring (Fig 4.16).


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Fig. 4.16 13C-NMR of DP-II

Based on above mentioned observations derived from various spectral data, DP- II was

assigned a chemical name 1-((1,3-dioxolan-2-yl)methyl)-N-methyl-4-(methylamino)-1H-

imidazole-5-carboxamide having molecular formula C10H16N4O3.


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Scheme 4.2 Fragmentation pattern of degradation products DP-I and DP-II


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Fig 4.17 Structure of a) Doxofylline, b) Degradation product I and c) Degradation product II

Table 4.1 Comparative 1H and 13C- NMR assignments for a) Doxofylline b) DP-I and c) DP-II

(DMSO-6)

s: singlet; d: doublet; m: multiplet.

- Disappearance of signals in degradation products when compared with that of doxofylline

Position of Carbon

(ppm) Dox (ppm) DP I (ppm) DP II C Dox 13C DP I 13C DP II

1N-(CH3) 3.17 (s, 3H) 3.20 (s,3H) 2.70 (d, 3H) 27.4 27.5 25.5 1N-H 7.08 (d, 1H)

2 150.8 150.9 - 3N-(CH3) 3.37 (s, 3H) 3.41 (s,3H) 2.75 (d, 3H) 29.3 29.4 30.2

3N-H - - 5.20 (d, 1H) - 4 147.9 148.1 138.3 5 100.6 106.0 101.2 6 154.4 154.5 161.6 7 8 7.97 (s, 1H) 7.93 (s, 1H) 7.34 (s, 1H) 143 143.2 151.9 9

Dioxalane ring

1’ 4.39 (d, 2H) 4.10 (d, 2H) 4.27 (d, 2H) 47.5 52.0 48.1 2’ 5.17 (t, 1H) 5.04 (t, 1H) 5.02 (t, 1H) 106.1 87.8 105.7 3’ 6.18 (d, 1H) - - - - 4’ 3.79 (m, 2H) - 3.70 (m, 2H) 64.5 - 64.4 5’ 3.82 (m, 2H) - 3.80 (m, 2H) 64.5 - 64.4 6’ 6.18 (d, 1H) -


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4.3.4 Plausible mechanism for formation of degradation products

4.3.4.1 Degradation Product-I (DP-I)

The mechanism is the reverse of acetal formation. The mechanism involves the protonation

of the oxygen atom in the dioxalane ring. Hydroxyl group gets eliminated as oxygen

becomes oxonium ion by the movement of lone pair of the electrons present on the oxygen

atom of the dioxalane ring. Water acts as a nucleophile and attacks the unsaturated carbon.

Elimination of the proton and the nucleophilic attack of the water molecule on the carbon

favor the removal of ethylene glycol molecule. Elimination of the proton from the water

molecule gives the product gem diols. Gem diols are not stable and it loses a molecule of

water to give rise to aldehydes. But aldehydes in highly protonated environment will be

hydrated and exist as gem diols. (Scheme 4.3)

4.3.4.2. Degradation Product-II (DP-II)

The pyrimidine ring of xanthine moiety which consists of two amide bond is susceptible to

base hydrolysis because of presence of electrophilic carbonyl carbon present in xanthine

ring system. In base hydrolysis, hydroxide ion attacks the electron deficient carbon and

forms the carboxylic acid. Hydroxide ion can either attack the C-2 or C-6 position

preferably attacks the C-2 position rather than C-6, since ureido moiety is more susceptible

to base hydrolysis than amide motif. In basic environment, formed carboxylic acid prefers

to exist as carboxylate anion and therefore acidic proton makes 1, 2 shift to the basic

nitrogen (either N-1 or N-3). This paves the way for elimination of carbon dioxide giving

rise to product. The final product is the same whether the bond breakage is between N-1

and C-2 or C-2 and N-3. (Scheme 4.3)


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Scheme 4.3 Plausible mechanism of formation of degradation products


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4.3.5. Validation of LC method

4.3.5.1. Specificity

The method proved to be specific to each peak, which was indicated through peak purity

data obtained using a photo diode array detector. The resolution between doxofylline and

its degradation products is greater than 1.5, indicating the method is specific for

doxofylline and its degradation products. (Fig. 4.1h, Table 4.2).

Table 4.2 Peak purity and resolution of drug and degradation product

Drug/Degradation product Peak purity index Resolution factor

I 0.9853 1.98

II 0.9996 1.69

Doxofylline 0.9981 7.41

4.3.5.2 Linearity

The linearity of the method was established between concentrations range between 5g

mL-1 to 30g mL-1. The r2 value was 0.998 as shown below

Table 4.3 Linearity study

Conc.

(g mL-1)

Area1 Area 2 Area 3 Average ± S.D ± R.S.D (%)

5 227122 227350 224027 226166 1856.22 0.82 10 428755 432151 422507 427804 4891.78 1.14 15 634149 636122 625012 631761 5927.47 0.93 20 851124 855239 839254 848539 8300.104 0.97 25 1014984 1024728 1000161 1013291 12370.69 1.22 30 1309719 1298263 1290024 1299335 9891.192 0.76

4.3.5.3 Precision

The % R.S.D. for intra- and inter-day precision studies at three different concentrations in

triplicate, viz., 10g mL−1, 20g mL−1 and 30g mL−1 was < 2%, signifying that the

method was sufficiently precise. (Table 4.4).


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Table 4.4 Intraday and Inter day precision study

Concentration (g mL-1) Intra Day Precision

Measured Conc. (g mL-1) ± S.D., R.S.D (%)

Inter Day Precision

Measured Conc. (g mL-1) ± S.D., R.S.D (%)

10 10.163±0.1457,1.43 10.166±0.1169,1.15

20 20.160±0.2523,1.25 20.176±0.1764,1.04

30 30.661±0.0873,0.28 30.886±0.2977,1.16

4.3.5.4 Accuracy

The percentage recovery of added drug ranged from 98.1 to 102.9 % w/w. (Table 4.5)

Table 4.5 Recovery data for drug spiked into a mixture of stressed samples

Spiked Conc. (g mL-1)

Calculated spiked Conc. (g mL-1) ± S.D., R.S.D (%)

Recovery (%)

10 9.81±0.2418,0.37 98.10

20 20.32±0.2135,0.62 101.60 30 30.87±0.4286,1.37 102.90

4.3.5.5 LOD and LOQ

The limit of detection and limit of quantitation was found to be 0.1 g mL-1 and 0.7 g

mL-1 respectively.

4.4 Summary

The present study revealed very important and useful information which has not yet

reported in the literature of doxofylline. The result of the present work can be summarized

as follows:

1. The drug was found to be susceptible to base hydrolysis followed by acid

hydrolysis and was stable in other stress conditions.

2. Identification and characterization of the degradation products by IR, mass and

NMR spectroscopic technique elucidate the structure of DP-I as

a. 7-(2, 2-dihydroxyethyl)-1, 3-dimethyl-1H-purine-2, 6(3H, 7H) – dione and

DP-II as


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b. 1-((1, 3-dioxolan-2-yl) methyl)-N-methyrl-4-(methylamino)-1H-imidazole-

5-carboxamide respectively.

3. Mechanistic pathway for formation of degradation products was laid down.

(title of the thesis)shodhganga.inflibnet.ac.in/bitstream/10603/9713/14/14-chapter 4.pdfdoxofylline,...

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