~sohtion and Characterization of

Degradan t Impurities in Dipyridamob Formulhtion

Dipyridamole is an antithrombotic drug. In the stability study of drug

product of dipyridamole, two unknown impurities (referred as DP-I and DP-11)

were detected at levels of 0.25% and 0.54% by gradient reverse phase HPI,C

method. The drug product was subjected to stress to enhance the level of these

impurities. An elegant isocratic preparative method was employed using a

Reprosil CN column with a short run time of 14 minutes to isolate these

impurities. The DP-I and DP-I1 were isolated with purities of 99.1% and 99.8%

respectively. Structural studies of these impurities were undertaken using

spectroscopic techniques such as IR, NMR and Mass. Based on the spectral data,

the structures of DP-I and DP-I1 have been characterised to be 2.2'.2",2"'-(4-

hydroxy-%(piperidin- 1 -yl) pyri111ido [5,4-dlpyrimidine-2,6 diyl) bis(azanetriyl)

tetraethanol, 4-(2-((6-(bjs (2-hydroxyethyl) amino)-4, 8-di (piperidin-1-yl)

pyrimido [ 5 , 4 4 1 pyrimidin-2-yl) (2-hydroxyethyl) amino) ethoxy)-2. 3-

dihydroxy-4-oxobutanoic acid, respectively. A detailcd elucidation of the structure

is presented in this article.

3. Introduction

Pharmacological studies on Dipyridamole, 2, 6-bis (dictha11olarnino)-4, 8-

dipiperidinopyrimido-[5, 4-dl pyrimidine suggest that it may be used as a

coronary vasodilator in clinical medicine. The known pharmacological approach

for the prevention of thrombotic accidents is to use Dipyridamole in combination

with Aspirin (acetylsalicylic acid). Dipyridamole is well accepted for treatment of

angina pectoris and cerebrovascular diseases. 'The mechanism of action is to

inhibit the cellular uptake and metabolism of adenosine with resulting

vasodilatory and anti-aggregatory effects. Through the combination of its

antiplatelet and vasodilator function, Dipyridamole probably improves tissue

perfusion [I]. It has also been reported that Dipyridamole possesses antioxidant

properties and attenuates the formation of reactive oxygen species in platelets and

endothelial cells [2] . Recent studies reveal that combination of a very low dose of

Prednisolone and Dipyridamole has exhibited significant synergistic effects in

human clinical trials [3]. The same was also observed in collagen and adjuvant

arthritic animal based on inhibition of additional inflammatory chemokine [ S ] . On

the contrary, Dipyridamole did not modify the symptoms of rheumatoid arthritis

C L p - I I I I m I m

in other clinical trial described by Forrest et al. [ 5 ] . In view of its wide use, the

stability indicating methods for the determination of degradation products in

Dipyridamole [6-81 have received much attention.

It is mandatory to identify and structurally characterise any impurity

formed during production and stability, exceeding the identification threshold of

0.2% in the drug product 19-1 51. For low-level impurities or degradation products,

this quite often involves H P I X method development, isolation and

characterisation techniques 116-1 91. This paper describes the separation, isolation

and characterisation of two unknown degradation impurities formed in

Dipyridamole.

OH I

14 \ 12 36

19

21 OH 22 30

12

OH 22 30

Dipyridamole DP-I DP-I1

Fig. 3a. Chemical structures of Dipyridamole and its degradant products DP-I

and DP-11

Fig. 3b. HPLC initial chromatogram of Dipyridamole sample

Fig. 3c. HPLC stability chromatogram of Dipyridamole

3.1. Experimental

3.1.1. Chemicals and reagents

Dipyridamole drug product used for investigation was obtained from the

R&D of Dr. Reddy's Laboratories Ltd., Hyderabad, India. HPLC grade

acetonitrile and methanol were obtained from the Merck Co., Mumbai, India.

Ultra pure water was collected from TKA Millipore water purification system.

Ammonium formate, formic acid, potassium dihydrogen phosphate, sodium

Cliapter-111 IC'D1D.F

hydroxide were procured from Rankem India Ltd. Chloroform-d.3. methanol-d4 and

dimethyl sulphoxide-d6 (for NMR) were purchased from Aldrich Chemical Co..

USA.

3.1.2. High performance liquid chromatography (analytical)

Samples were analysed on Agilent HPLC-DAD system equipped with

1100 series low pressure quaternary gradient pump along with pulse dampener.

AA lnertsil ODs-2 column (Agilent 150 x 4.6 mm. 5pm) was used for

chromatographic separations. Mobile phase A consists of a mixture of 0.01 M

KH2P04 buffer, with the pH adjusted to 7.0 using 5% sodium hydroxide, and

methanol in the ratio of 50:50 (viv). Mobile phase B consists of methanol and 0.01

M KI-IzPOd buffer in the ratio of 95:s (vlv). The following gradient prvgramrne is

employcd: 'r (min)/%R (v/v): 0i0, 410, 25/100, 28/100. 30/0, 35/0. The sample

detection was monitored at a wavelength of 295 nm. Chromatography was

performed at 45OC using a flow rate of 1 rnllmin.

3.1.3. Forced degradation of Dipyridarnole drug product

Forced degradation studies were performed on the Dipyridmole drug

product with the intention of determining the conditions responsible for the

formation of the degradation products. Accordingly, degradation studies were

conducted by stressing with acid, base, peroxide and thermal.

3.1.3.1. Acid stressed degradation

200 rng equivalent of Dipyridamole drug product was dissolved in 20 mL

of methanol and water in the ratio of 1 : 1. HCI solution (5 mI, of 0.1 N) was added

and refluxed for .about 4 hours and neutralised.

Fig. 3.1.3.1. Typical chromatogram of Dipyridamole acid degradation

3.1.3.2. Base stressed degradation

200 mg cquivalcnt of Dipyridamolc drug product was dissolved in 20 ml, o f

methanol and water in the ratio o r 1: 1 . NaOH solution (5 rnl, o f 0. IN) was added and

refluxed for about 4 hours and neutralised.

Fig. 3.1.3.2. Typical chromatogram of Dipyridamole base degradation

3.1.3.3. Peroxide stressed degradation

200 mg equivalent of Dipyridamole drug product was dissolved in 20 mL of

methanol and water in the ratio of 1 : l . Hydrogen peroxide (5 mL of 6 % solution)

was added and maintained at 70°C for 4 hours.

Fig. 3.1.3.3. Typical chromatogram of Dipyridamole peroxide degradation

3.1.3.4. Thermal stressed degradation

200 mg equivalent of Dipyridamole drug product was taken in a Pctri dish.

Water was sprinkled on the drug product and subjected to 105'C for 3 hours.

Fig. 3.1.3.4a. Typical chromatogram of Dipyridamole thermal degradation

Fig. 3.1.3.4b. Overlay of HPLC chromatograms of Dipyridamole forced

degradation studies.

3.1.4. HPLC analyses of forced degradation samples

The degradation samples were analysed by HYLC method as described in

section 2.2. Under acid stressed conditions, UP-I and UP-11 have formed upto

3.0% and 0.4 %, while peroxide degradation yielded 0.6% and 1.1 %,

respectively. In thermal stressed condition, only DP-I1 was formed upto 7.7%. In

base stressed condition. there was no significant degradation. Hence the thermal

and acid degradation routes were chosen to enhance the impurities.

3.1.5. Sample preparation

Dipyridamole sample was prepared at a concentration of 1 mgJm1, in

methanol for the analytical HPLC and 50 mg/ml, for the preparative HPLC

analyses.

3.1.6. High performance liquid chromatography (preparative)

Impurities were isolated from the sample using Gilson GX281 preparative

HPLC Binary system equipped with a Gilson photodiode array detector model

172 along with a Gilson analytical HPLC system. Data was collected and

processed using TRILUTION LC software (vcr 2.1). Approximately 50 mg/mL of

sample was prepared to load on to the column. A Reprosil CN column (250 x 20

Ckpttr-III I m I m

mm, 5 p ) was employed for the separation of DP-I and DP-11. The mobile phase

consists of a mixture of 0.02M ammonium acetate buffer, with the pH adjusted to

7.0 with ammonia and acetonitrile in the ratio of 70:30. The flow rate was kept at

15 mWmin. Detection was carried out at 295 nm. Desaltification procedure was

used to remove the buffering agents leading to pure fractions of the impuritics.

The impurity fractions from several injections were pooled. The pooled fraction

w& concentrated by using Rotavapnur (Heidolph Laboratory 4002 control) under

high vacuum. The aqueous solution was lyophilized to solidify the impurities.

Fig. 3.1.6a. Preparative chromatograms of DP-I & DP-I1

om m tm sm sm ~osl lm fim i sm rm am nm nm am am am nm um Yllrr

Fig. 3.1.6b. HPLC Purity Chromatogram of DP-I

Fig. 3.1.6~. HPLC Purity Chromatogram of DP-I1

3.1.7. Mass spectrometry (LC-MS/MS)

LC-MS/MS analyses has been performed on API 4000 model Mass

Spectrometre (Applied Bio systems). Analyses was pcrformcd in positive.

ionisation mode with turbo ion spray interface. Thc parametrcs for Ion source

voltage (IS) = 5500 V, Declustering Potential (I3P) = 70 V, Entrance Potential

(EP) = 10 V were set with nebulizer gas as air at a pressure of 40 psi and curtain

gas as nitrogen at a pressure of 25 psi. An lnertsil C8 column (250 x 4.6mm, 5pm)

was used for the separation. Mobile phase A is a mixture, consisting of 0.01 M

Ammonium formate buffer with the pIl adjusted to 4.0 using formic acid and

acetonitrile in the ratio of 70:30. Mobile phase B is a mixture consisting of above

buffer and acetonitrile in the ratio of 55:45. The gradient conditions employed for

the separation with a timed gradient programme of T (min)/%B (vlv): 0 /0,

40/100,41/0,45/0. The flow rate was kept at 1 rnllrnin.

Fig. 3.1.7a. LC-MS data of Dipyridamolc

Fig. 3.1.7b. LC-MS data of DP-I

Fig. 3.1.7~. LC-MS data of DP-11

Table 3.1.7. Fragmentation pathways and infrared data of Dipyridamole, DP-

I and DP-I1

Name

3.1.8. UPLC-TOF-MS

The UPLC-TOF-MS system consisted of an ACQUITY'"' MJltra

Performance Liquid Chromatography system and a Micro mass LCT Premier XE

Mass Spectrometre (High sensitivity orthogonal time-of-flight instrument, Waters,

Millford, USA) equipped with a lock mass sprayer, operating in either the positive

or negative ion mode. All analyses were acquired using the lock spray to ensure

Fragmentation Pathways

accuracy and reproducibility; leucine-enkephalin was used as the lock mass.

Sample of concentration 0.02 mg/mL in methanol was infused in TOF-MS at a

FT-IR

(cm-I)

Cliapur-III 1rnIQ)cI:

tlow rate of 10 pL/min. High resolution (W mode. FW.HM 10500) positive

polarity scan responses were collected from rn/z 100 to 1000 at a rate of 1.0

slscan.

Singk Mur A M C I S T o l a 8 n e a = S O P P ~ I DBE m n = 0 0 r n a r = ~ ) o E b m n l pedrtm MI Nwnterdsoiopa~rmdlor lorFlT=3

Mmdsobp. kbs, E m Ek,m lrm 3B W a l e ) e n m a w mm 7 rauns wmlunb (up b 4 M oaW M a s ta ubl m1.1 E r n UICd C 0-35 H 0 35 N a9 0 0.10 I U ~ W * u o a b - w r - . w 2ahalorr

1 TWUSESt "Ml, J I 5 , " Is i ,cmg, , l . lYOl l ,

.a2451 Ilh.UO1

Im

X ' m2u1 11?W

Im!m ,$$,iW,602,, JIIW, )62Ql%.mam u o 2 x u . . - . .... 3:Y:05Y. . E . % 9 . e Y g 611 0% .r?!?!!lb'E- (ml!R!Y)!II*IUq* 1'1~*1). wz

I Y ~ z ~ w s o u ) ~ ~ ~ u o I D ~ ~ I Q D J O ~ ~ W ~ W ~ ~ ~ . ~ ~ ~ ~

*Llili.i.

*.Xlnur 0 0

5 0 I U 8 0 0

*as. C . I C * I " . * P V * O.. , F I T $>?mu,.

4 5 2 6 I 2 I I I rl 0 C19 1112 N I 0 ,

' i l ill3 ) 0 t d 5 7- I CJO " $ 2 H 01

Fig. 3.1.8a. HRMS data o f DP-I

Slnglo Mars Analyris Tderance*SOPPhl I DEE mn=OO max=800 Etnnent @ran on ~ d 1 s d c $ e p i * r ~ b l F I T : 3

LlnPhQOprHUt EmEeemkm n s b m ( a ) a ~ b s d m z ~ m ~ m ~ ( u p b ~ b s r ~ ~ f ~ ~ * c h - ~ E h n * Usad Cb35 Hn3 Nbe O b i 0 IM *ub rokmmr -w ~ ~ O I I

I TWUFES* W l l J2IiUIIO)Cmll)

631 311P 2 !&.on

':I I

e l o n 1 3 m m

i Gum .k-mfr- - .I ., - j ~ w , ..to ~ 3 1 ? ~ T a ' 1 ' ~ - ) a * I - - - ~ Y ? t aim?rn ysoII ~ s ~ ~ ~ ~ ~ m ~ ~ ~ ~ ~ l i ~ ~ ~ ~ ~ d ~ w ~ r n s n * o s m * o ~ r m r m

Hinlsro 0 0 mi- 5 0 5 0 8 0 0

MI C ~ C m . ~ dl m DU i PIT ro-1.

631 1119 617 1110 1 9 I 0 1 5 1 S C Z l Nb5 ## W 617 1150 I 1 I 1 14 5 2 I C31 H O U6 07

Fig. 3.1.8b. HRMS data of DP-11

3.1.9. NMR spectroscopy

NMR experiments were performed on 400 MHz Mercury plus NMR

spectrornetrc (Varian) in DMSO-d6+CDC13 and Methanol-d4 at 2S0 C. Proton and

carbon chemical shifts were reported on S scale in ppm, relative to TMS (6 = 0.00

ppm), CDC13 (S = 77.00 ppm), CD30D (6 = 49.1 pprn) and DMSO (6 = 39.50

pp.m) as internal standards. Standard pulse sequences provided by Varian were

used for 'kf NMR, gradient Double Quantum - filtered Correlation Spectroscopy

(gDQCOSY), gradient Heteronuclear Single Quantum Coherence spectroscopy

(gHSQC) and gradient Heteronuclear Multiple Bond Correlation spectroscopy

(gHMBC).

Fig. 3.1.9b. Proton decoupled I3c NMR spectrum of DP-I.

Fig. 3.1.9~. 'H NMR spectrum of DP-11

Fig. 3.1.9d. Proton decoupled 13c NMR spectrum of DP-I1

Fig. 3.1.9e. gDQCOSY spectrum of DP-I

Fig. 3.1.9f. gDQCOSY spectrum of DP-I1

Fig. 3.1.9g. gHMBC spectrum of DP-I1

- Dipyridamole DP-I DP-I1

Positiona 'H I3c 'H I3c 'H 13c

1 153.2 (C) 146.7 (C) 153.2 (C)

3 159.3 (C) 160.5 (C) 159.3 (C)

4 131.9 (C) 128.0 (C) 132.1 (C)

5 131.9 (C) 128.0 (C) 131.8 (C)

8 153.2 (C) 146.7 (C) 152.8 (C)

10 159.3 (C) 158.2 (C) 159.3 (C)

5 1.6 (CH2)

5 1.2 (CH2)

64.0 (Cl12)

59.5 (CI-12)

47.6 (CH2)

24.5 (CH2)

25.8 (CH2)

5 1.2 (C1-12)

51.2 (CHI)

59.5 (CHz)

59.5 (C'112)

47.9 (CH2)

24.5 (CH2)

25.8 (CH2)

47.9 (CH2)

172.1 (C=O)

72.4 (CH)

72.2 (CH)

173.0 (C=O)

Table 3.1.9. 'H and ''c NMR assignments for Dipyridamole, DP- I and DP-I1

a Refer structural formula (Fig. 1 (A)) for numbering: s, singlet; d, doublet; t, triplet; m, multiple; The correlations shown in the 'H and 13c NMR columns are in agreement with t1SQC and HMBC

spectra.

ctiap-111 IrnIQM:

3.1.10. FT-IR spectroscopy

IR spectra were recorded in solid state as KBr dispersion medium using

Perkin-Elmer FT-IR spectrophotometre.

Fig3.l.lOa. IR Spectrum of Dipyridamole

44.

W

48.

Y.

Y.

Y.

?Mi IU YOD f3P Y lOW U _ . I 1 1 U )1 IQ I U QO

Fig.3.l.lOb. IR Spectrum of DP-I

39.mJ - * I r I t . r t - 1- - - - POW 9f00 moo asPo 2000 moo - -7

low en* vm

Fig.3.1.10~. I R Spectrum of DP-11

3.2. Results and discussion

3.2.1. Analytical HPLC of drug product

A typical analytical HPLC chromatogram of a stability batch of

Dipyridarnole drug product recorded using the HPLC method as described in

Section 2.2 is shown in Fig. l (B). DP-I, DP-11 and Dipyridamole elute at retention

times 3.4, 10.7 and 16.8 min respectively while Imp -1, 2, 3 and 4 are known

impurities. The 'representative HPLC chromatograms of the forced degradation

study are shown in Fig. 1 (C). The structures of these impurities and Dipyridarnole

are shown in Fig.1 (A). Both DP-I and DP-I1 are more polar with respect to

Dipyridamole.

3.2.2. Isolation of impurities by preparative HPLC

isolation of these impurities by preparative HPLC was a challenging job as

the two target impurities were eluting very closely to the parent peak in

preliminary studies. This was due to limitations of preparative chromatography.

which is traditionally associated with low efficiency columns, packed with lcss

pure silica (compared with analytical columns), unstable and packed with

conventional packing technologies.

With relatively high particle size (7-10 pm) of the stationary phase used in

preparative HPLC columns, it is indccd a challenging task to resolve the peaks of

the target impurities from the main peak despite the maximum injection load and

yet leading to isolation of impurities in sufficient quantities with high purity.

Several trials were performed to achieve the required resolution. Finally an

isocratic solvent system was developed with good resolution and a short runtime

(14 min). The purity of DP-I and DP-I1 were 99.1 and 99.8% rcspcctively and

these samples were used for spectroscopic structural studies.

3.2.3. Structure elucidation of Dipyridamole DP- I

In the positive and negative mass spectra, the protonated and deprotonated,

[M+H] ' and [M-HI-, molecular ions were detected at d z 438.1 and 436.4,

respectively. The evcn m/z number of [M+II] ' and [M+kl] * ions suggest that DP-

I contains odd number of nitrogen atoms (nitrogen rule). From these results the

molecular ion of DP-I was found lo be at mlz 437. The mass difference between

Dipyridamole and DP-I was found to be 68 amu.

The positive HR-MS spectrum showed protonated molecular ion at m h

438.2453 corresponding to molecular formula C19k132N705 Whcn compared with

the molecular formula of Dipyridamole, there was a difference of Cs119N and an

addition of one oxygen atom. The differencc can bc rationalised in terms of the

loss of piperidine moiety and an addition of one hydroxyl group. The LC-MS data

of DP-I is shown in Fig.2 (A).

cliap-I11 I m I W

The proton NMR spectra of Dipyridarnole and DP-1 showed three sets of

signals. The 'H NMR and I3c NMR spectra of DP-I are shown in Fig.3 (A) and

(B), respectively. The proton signals in DP-I were observed at 4.02 (4H). 3.6-3.7

(16H) and 1.63 ppm (6H), where as Dipyridamole showed signals at 4.1 (8H), 3.7-

3.8 (16H) and 1.7 (12H) protons and the exchangeable protons were not observed.

From the gDQCOSY data, it was observed that the protons at 4.02 ppm showed

correlations with the proton at 1.63 ppm. These can be attributed to piperidine

moiety. Furthcr, the protons at 3.6 ppm showed correlations with the protons at

3.7 ppm corrcsponding to methylazanediyl dicthanol moiety. In DP-I. the protons

corresponding to piperidine were found to be ten, confirming that this is only one

piperidine moiety. The remaining protons from methylw.ancdiyl diethanol

corresponding to sixteen were found to be intact.

The absence of piperidine group shows that there are 8 4 mass units less

(CsHloN) when compared to Dipyridamole. Rut the MS data showed that there is

only a 68 mass unit difference. This shows that there is an addition of 16 mass

units after the removal of piperidine moiety. This observation indicates that the

piperidine moiety was replaced by a hydroxyl group.

The above spectral data supports the assigned structure as 2,2',2".2"'-(4-

hydroxy-8-(piperidin-1-yl)pyrimido[5,4-d]pyrimidine-2.0 diyl) bis(az.anetriy1)

tetraethanol (DP-I).

3.2.4. Structure elucidation of Dipyridarnole DP- 11

In the positive and negative mass spectra, the protonated [M+H] ' and

deprotonated . [M-HI- molecular ions were detected at m/z 637.3 and 635.7

respectively. From these results the molecular ion of DP-I1 was found to be at m/z

636. The mass difference between Dipyridamole and DP-11 was found to be 132

arnu. The ES-MS-MS spectrum displayed product ions at m/z 619.6, 591.5, 561.4,

505.2, 487.3 and 404.3 in which 505.2 is the dominant fragment. The fragment

ion at 505.2 suggests that the parent moiety is intact in DP-11.

Cliapter-111 ImIQ)F

The positive HR-MS spectrum showed protonated molecular ion at m/z

637.3329 corresponding to molecular formula CzsHjsN809. When compared with

the molecular formula of Dipyridamole, there was an addition of C4I-14o5. The

LC-MS data of DP-I1 is shown in Fig.2 (B).

The 'H NMR and I3c NMR spectra nf DP-I1 are shown in Fig.4 (A) and

(B), respectively. On comparison, DP-I1 showed two additional protons at 4.5

(1H) and 4.32 (IH) as doublets and their corresponding carbon signals were

observed at 72.4 and 72.2 ppm. respectively. From the proton and carbon

chemical shifts thcse can be attributed to oxygen attached methine groups. The 13 C NMR spectrum showed two carbonyl signals at 173.0 and 172.1 ppm. The

protons at 4.5 and 4.32 pprn showed MMBC correlations to the carbon signals at

173.0 pprn and 172.1 pprn respectively. From thcse observations the additional

moiety can be attributed to tartaric acid group, which was found to be one of the

excipients used in the formulation. Thc additional carbonyl groups wcre further

confirmed from IR spectroscopy at 1741 cm-I. Interestingly one of the carbonyls

at 172.1 pprn showed additional cross peak with one of the mcthylcne protons

(4.45 pprn as a triplet) of methylazanediyl dirthanol of Dipyridamole.

The above spectral data supports the assigned structure as 4-(2-((6-(bis (2-

hydroxyethyl) amino)-4, 8-di (piperidin-1-yl) pyrimido [5, 4-d] pyrimidin-2-yl)

(2-hydroxyethyl) ammo) ethoxy)-2,3-dihydroxy-4-oxobutanoic acid (I>P 11).

3.3. Conclusion

The drug product of Dipyridamole, subjected to stability studies, was

evaluated for degradation products. In addition to pharmacopoeia impurities, two

unknown impurities which are not reported in the literature were observed. Stress

study was performed to enhance the impurities. These degradation products were

isolated by a simple isocratic method and characterised by IR, NMR, MS and

HRMS spectroscopic techniques. The most probable structures are proposed for

these impurities based on the spectral data.

3.4. References

N.P. Stafford, A.E. Pink, A.E. White. J.R. Glenn, S. lieptinstall,

Mechanisms involved in Adenosine 'i'riphosphate-Induced platelet

aggregation in whole blood. Arterioscler. Thromb. Vasc. Biol. 23 (2003)

1928-1933.

S. Chakrabarti. 0. Vitseva, D. Iyu, S. Varghese, J.E. Freedman, The effect

of Dipyridarnole on vascular cell-derived reactive oxygen spccies. J.

Pharmacol. Exp. Ther. 3 15 (2005) 494-500.

T.K. Kvien, E. Fjeld. B. Slatkowsky-Christensen, M. Nichols, Y. Zhang,

A. Proven, et al, Efficacy and safety of a novel synergistic drug candidate,

CRx-102, in hand osteoarthritis, Ann. Rheum. Dis. 67 (2008) 942-948.

G.R. Zimmermann, W. Avery, A.1,. Finelli, M. Farwell. C.C. Frascr, A.A.

Borisy, Sclectivc amplification of glucocorticoid anti-intlammatory

activity through synergistic multi-target action of a combination drug,

Arthritis. Res. Ther. 1 1 (2009) 105.

C.M. Forrest. T.W. Stone, G.M. Mackay, I,. Oxford, N. Stoy, G. Ilarman

et al, Purine metabolism and clinical status of patients with rheumatoid

arthritis treated with Dipyridamole, Nuclcosides Nuclcotides Nucleic

Acids. 25 (2006) 1287-1 290.

J. Zhang, R. B. Miller, R. Jacobus, Development and validation of a

stability-indicating HPLC method for the determination of degradation

products in Dipyridamole injection, Chromatographia. 44 (1 997) 247-252.

K. Prakash, Rama Rao Kalakuntla, Jayapal Reddy Sama, Rapid and

simultaneous determination of aspirin and Dipyridamole in pharmaceutical

formulations by reversed-phase high performance liquid chromatography

(RP-HPLC) method, Afr. J. Pharm. Pharacol. S(2) (201 1) 244-25 1 .

J.H. Bridle, M.T. Brimble, A stability indicating method for Dipyridamole,

Drug. Dev. Ind. Pharm. 19 (3), (1993), 371 -381.

International Conference on Harmonisation of technical requirements for

registration of pharmaceuticals for human use, ICH Q3A (R2), Impurities

in New Drug substances, 2006, pp. 1 - 15.

CIi~ptcr-I11 IWIDQ

10. International Conference on Harmonisation of technical requirements for

registration of pharmaceuticals for human use. ICH Q3B (R2). Impurities

in New Drug products. 2006, pp. 1-16.

11. International Conference on Harmonisation of technical requirements for

registration of pharmaceuticals for human use, ICH Q I A (R2) Stability

testing of new drug substances and P~oducts. 2003. pp. 1-24.

12. L7.S. Food and Drug Administration. Drug Stability Guidelines. February

1987.

13. S. Gordg, New safe medicines faster: the role of analytical chemistry.

Trends in Anal. Chem. 22 (2003) 407.

14. J.A. Mollica, S. Ahuja, Cohen, Stability of Pharmaceuticals. J. J. Pharm.

Sci. 67, (1978) 443.

15. l h e United States Pharmacopeia. 25th Ed.. Ilipyridamole. llnited States

Pharmacopeial Convention, Inc., Rockville. MD, 'IJSA. 2000. p. 7.

16. Satindcr Ahuja, Karen Mills Alsante, Handbook of isolation and

characterisation of impurities in pharmaceuticals, first ed.. USA. 2003.

17. R.J. Smith, M.I.. Webb, Analyses of Drug Impurities, Blackwell

Publishing, first ed., UK, 2007.

18. S. Ahuja, Impurities Evaluation of Pharmaceuticals, Marcel Dekker, first

ed., NY, 1998.

19. Budzikiewic7. Herbert, Djerassi. Carl, Dudley H. Williams, Mass

spectrometry of organic compounds, Holden-day, Inc., first ed., California,

1967.