DSpace at VNU: Effects of Solvents and Crystallization Conditions on the Polymorphic Behaviors and Dissolution Rates of Valsartan

Samples were prepared by recrystallization from water or organic solvents, such as acetonitrile, acetone and ethanol, using methods with and without heating.. Method 1:1 g of VAL was dis

Effects of Solvents and Crystallization Conditions on the

Polymorphic Behaviors and Dissolution Rates of Valsartan

Thao Truong-Dinh Tran 1,2 , Phuong Ha-Lien Tran 1,2 , Jun-Bom Park 2 , and Beom-Jin Lee 2,3

1 Biomedical Engineering Department, International University-Vietnam National Universities, Ho Chi Minh City, Vietnam,

2 Bioavailability Control Laboratory, College of Pharmacy, Kangwon National University, Chuncheon 200-701, Korea, and

3 College of Pharmacy, Ajou University, Suwon 443-749, Korea

(Received January 3, 2012/Revised April 1, 2012/Accepted April 4, 2012)

For the quality evaluation of raw materials, the influence of various types of solvents on the

polymorphic crystallization behaviors and dissolution rates of two sources of valsartan (VAL)

from China and India was investigated Samples were prepared by recrystallization from

water or organic solvents, such as acetonitrile, acetone and ethanol, using methods with and

without heating Recrystallization behaviors were characterized by differential scanning

calo-rimetry (DSC) and powder X-ray diffraction (PXRD) Scanning electron microscopy (SEM) was

also used to observe the morphology of samples The dissolution rate of recrystallized samples

in water was evaluated and compared to the original VAL sample There were significant

dif-ferences in morphology, crystal structure and dissolution rate among the samples

recrystal-lized using organic solvents VAL was transformed into another polymorphic form by the

solvents and recrystallization conditions These physical properties of VAL also differed

between the two sources of VAL Thus, the physicochemical differences of raw materials

should be carefully considered in early dosage formulation approaches

Key words: Valsartan, Crystallization condition, Structural behavior, Solvent type,

Polymor-phism, Dissolution rate

INTRODUCTION

It is known that pharmaceutical compounds can exist

in more than one crystalline form, each of which can have

different physicochemical properties This phenomenon

is called polymorphism (Näther et al., 2002; Suitchmezian

et al., 2006; Barone et al., 2011; Remko et al., 2011;

Mandal et al., 2012) These changeable behaviors can

impact the solubility, dissolution, bioavailability and

manufacturability of the drug product (Singhal and

Curatolo, 2004; Pilcer et al., 2012) Thus, the

physico-chemical quality of various raw materials should be

fully evaluated

Factors commonly cited that affect the crystal

forma-tion of pharmaceutical compounds include

supersatur-ation, agitation rate, cooling rate, solvent composition,

temperature, seed crystals, additives and impurities (Kitamura and Ishizu, 1998; Li et al., 1999; Shekunov and York, 2000; Shan et al., 2002) Among these factors, the solvent is considered to have a strong influence on the crystalline structure (Femi-Oyewo and Spring, 1994; Horst et al., 2001) Hence, solvent screening is

of foremost importance for the pharmaceutical industry

to avoid the unexpected appearance of a polymorphic form of a drug, which may lead to serious pharmaceuti-cal consequences resulting in delay of drug production Solid polymorphism of drug substances, therefore, has received much scrutiny in the development of dosage forms of pharmaceutical products

Valsartan (VAL) (Fig 1), N-(1-oxopentyl)-N-[[2'-(1H-tetrazol-5-yl) [1,1'-bi-phenyl]-4-yl]methyl]-L-valine, is

a selective angiotensin II type 1 receptor blocker indi-cated for the treatment of hypertension (Krishnaiah et al., 2010) The absolute bioavailability of oral VAL has been reported to be low because VAL belongs to the class II category according to the Biopharmaceutics Classification System (i.e., water-insoluble and highly

Correspondence to: Beom-Jin Lee, College of Pharmacy, Ajou

University, Suwon 443-749, Korea

Tel: 82-31-219-3442, Fax: 82-31-212-3653

E-mail: beomjinlee@gmail.com

permeable) (Cappello et al., 2006; Kumar et al., 2009;

Iqbal et al., 2010) For this reason, the crystal

modifi-cation of VAL has been undertaken to increase

dissol-ution and bioavailability (Park et al., 2010; Tapas et

al., 2010) However, the preparation and

manufactur-ing process of solid VAL may be complicated due to its

changeable crystallinity under different solvents and

manufacturing conditions.Thus far, no detailed

poly-morphic behaviors of VAL have been investigated

The objective of this study was to investigate the

effect of different solvents and crystallization

condi-tions on the structural behaviors of VAL To achieve this

aim, VAL was recrystallized from four types of solvents

(acetonitrile, acetone, ethanol and water) with and

without heating A dissolution test, differential scanning

calorimetry (DSC), powder X-ray diffraction (PXRD)

and scanning electron microscopy (SEM) were then

used to evaluate and identify crystalline structure of

the samples

MATERIALS AND METHODS

Materials

Valsartan (VAL) was obtained from Du-Hope

Pharma-ceutical Corporation and Jubliant Organosys Ltd Acetone

was purchased from Showa Ethanol was supplied by

Duksan Reagent and Chemicals Acetonitrile was

obtained from Fisher Scientific Korea Ltd Potassium

dihydrogen phosphate was purchased from

Sigma-Aldrich All other chemicals were of analytical grade

and were used without further purification

Recrystallization of VAL

Two different methods based on different

tempera-tures were used in this study to observe the effects of

solvents and crystallization conditions on the

struc-ture of VAL Method 1:1 g of VAL was dissolved in 10

mL of solvent (acetonitrile, ethanol, acetone or water)

at room temperature with stirring for 15 min, after

which the resultant solution (or suspension) was

im-mediately transferred to a freezer at −70oC for 3 h

Finally,the samples were dried in a vacuum dryer

Method 2: The same method as outlined in method 1

was used, with the exception that the solution was stirred at 50ºC for 15 min

HPLC analysis

VAL concentration was determined by an HPLC system (Waters) with a Luna 5 µ C18 analytical column (150 × 4.6 mm) A mobile phase of 0.015 M potassium dihydrogen phosphate (pH 2) and acetonitrile (55:45 v/ v) was used at a flow rate of 1.2 mL/min The UV de-tector was set at 234 nm to analyze the column effluent The entire solution was filtered through a 0.45-µm membrane filter (Millipore Corp.) and degassed prior

to use Each sample (20 µL) was injected into the HPLC system for analysis

SEM morphology

SEM was used to characterize the surface morph-ology of powders The samples were examined using a Cambridge Stereo Scan 200 at an accelerating voltage

of 15 kV The samples were mounted onto brass stages using double-sided adhesive tape and coated with gold-palladium for 60 seconds under an argon atmosphere using a Jeol JPC-1100 sputter coater (Jeol)

DSC behaviors

The thermograms of pure VAL and recrystallized powders were analyzed by DSC (TA Instruments, Model 2910) An approximate sample (0.4-0.5 mg) was weighed in a standard open aluminum pan An empty pan of the same type was used as a reference Dry nitrogen was used as the purge gas The samples were heated from 20 to 200oC at a heating rate of 5 oC/min The calibration of temperature and heat flow was performed with indium

PXRD patterns

VAL crystallinity was also investigated by PXRD The samples were scanned in steps of 0.02o from 5o to

60o (diffraction angle 2θ) at a rate of 1 second per step, using a zero background sample holder through a D5005 diffractometer (Bruker) using Cu-K radiation

at a voltage of 40 kV, 50 mA

Dissolution studies

Dissolution tests were performed with a DST-810 dissolution tester (Labfine) in 900 mL of water at 37oC using the paddle method at a rotation speed of 50 rpm Eighty mg of VAL powder was exposed to dissolution media without adding any excipients At determined time intervals, samples were withdrawn and replaced with an equal volume of dissolution media The drug concentration was then determined by the HPLC method as mentioned above

Fig 1 Chemical structure of valsartan.

RESULTS AND DISCUSSION

Structural behaviors

Thermal analysis (DSC)

Thermal analysis distinguishes polymorphs on the

basis of the phase transitions that occur during

heat-ing Therefore, we first analyzed the crystalline

struc-ture of VAL by using DSC to investigate the melting

point of the recrystallization samples The thermal

behaviors of different crystalline states of VAL are

shown in Fig 2 and Fig 3 The original VAL had a

dis-tinct melting peak at 91oC and 102oC for the materials

from China (Fig 2) and India (Fig 3), respectively

However, the way in which structural behaviors changed

depended on the type of solvent and recrystallization

conditions used Specifically, for the material from

China that had undergone method 1 (recrystallization

without heating), all the melting peaks of VAL were

shifted to the left: 68oC and 88oC (water); 37oC (ethanol);

51oC (acetone); 62oC and 87oC (acetonitrile) Similarly,

for the material from China that had undergone method

2, the melting peaks were observed at 67oC (water),

66oC (ethanol), 61oC (acetone), and 65oC and 90oC (ace-tonitrile) In addition, the material from India showed melting peaks for water, ethanol, acetone and acetoni-trile at 64oC, 56oC, 61oC and 62oC (method 1), and

65oC, 38oC, 56oC and 64oC (method 2), respectively These results indicate that VAL was recrystallized with different crystalline structures that were highly dependent on the source of material or recrystallization conditions (solvent and heating) (Megarry et al., 2011)

PXRD patterns

It is known that PXRD is another powerful technique suited for distinguishing solid phases for the identifica-tion of different internal crystalline structures There-fore, this technique was used to establish the differ-ences among crystal forms of the samples that were observed by DSC PXRD diffractograms of various recrystallization samples for the materials from China and India are shown in Fig 4 and Fig 5, respectively VAL had slightly broad peaks at 6.36o, 13.73o and

Fig 2 DSC thermograms of pure VAL (China source) and

its recrystallized powders by the two methods; Method 1:

VAL was dissolved in acetonitrile, ethanol, acetone or water

at room temperature; Method 2: VAL was dissolved in

ace-tonitrile, ethanol, acetone or water at 50 o C.

Fig 3 DSC thermograms of pure VAL (India source) and

its recrystallized powders by the two methods; Method 1: VAL was dissolved in acetonitrile, ethanol, acetone or water

at room temperature; Method 2: VAL was dissolved in ace-tonitrile, ethanol, acetone or water at 50 o C.

21.58o When VAL was recrystallized from the solvents,

the peak at 6.36o disappeared, except for the samples

recrystallized from acetonitrile These data indicate

that the crystallinity of VAL was partially changed to

an amorphous state (Hu et al., 2003) In contrast,

there are many specific peaks appearing from 8o to 23o

with the sample recrystallized from acetonitrile using

method 1, suggesting thatthe VAL structure became

more crystalline because these peaks did not exist for

VAL prior to recrystallization with acetonitrile In

contrast to the results using method 1, VAL showed

the same diffractogram as the original VAL when it

was recrystallized with acetonitrile using method 2

Meanwhile, as shown in Fig 5, the VAL from India

had a similar diffractogram to the VAL from China

However, when VAL was recrystallized by either method

1 or method 2, the diffractograms always showed

dis-appearance of the peak at 6.36o This result could suggest

that the VAL structure changed to an amorphous

state partially after recrystallization (Widjaja et al., 2011)

In summary, the appearance or disappearance of characterized peaks after recrystallization proved that

a polymorphic modification was obtained However, there are slight changes of VAL in amorphous formex-cept forthe VAL from China, recrystallized by method

1 with the use of acetonitrile Therefore, depending on the type of solvent and the source of drug material used

in the recrystallization, the drug formulation would be significantly affected by polymorphic behaviors

Morphology of VAL powder

The modification of crystalline structure may change the morphology of VAL Therefore, scanning electron microscopy (SEM) was used to observe the morphology

of the samples Fig 6 shows the morphology and shape

of untreated and recrystallized VAL (sourced from China) from various solvents It was observed that untreated VAL had a small irregular crystal shape, some of whichwere shapedlike needles In contrast, recrystallization of VAL from solvents produced a slab

of crystals Generally, method 2 produced a smoother

Fig 5 PXRD patterns pure VAL (India source) and its

recrystallized powders by the two methods; Method 1: VAL was dissolved in acetonitrile, ethanol, acetone or water at room temperature; Method 2: VAL was dissolved in acetonitrile, ethanol, acetone or water at 50 o C.

Fig 4 PXRD patterns pure VAL (China source) and its

recrystallized powders by the two methods; Method 1: VAL

was dissolved in acetonitrile, ethanol, acetone or water at room

temperature; Method 2: VAL was dissolved in acetonitrile,

ethanol, acetone or water at 50 o C.

VAL surface than compared to that of method 1 In

particular, some drug particles clearly had adsorbed

on the slabs recrystallized through method 1 in the

cases for water and ethanol Similar to VAL from China,

untreated VAL from India also showed small irregular

crystal shapes as shown in Fig 7 The morphology of the samples was smoother after treatment than com-pared with untreated VAL However, there was no dif-ference between method 1 and 2 after recrystallization Therefore, the morphology of VAL after

recrystalliza-Fig 7 SEM images of pure VAL (India source) and its

recrystallized powders by the two methods: pure VAL (A); VAL from acetonitrile using method 1 (B) and method 2 (C); VAL from ethanol using method 1 (D) and method 2 (E); VAL from acetone using method 1 (F) and method 2 (G); VAL from water using method 1 (H) and method 2 (I).

Fig 6 SEM images of pure VAL (China source) and its

recrystallized powders by the two methods: pure VAL (A);

VAL from acetonitrile using method 1 (B) and method 2 (C);

VAL from ethanol using method 1 (D) and method 2 (E);

VAL from acetone using method 1 (F) and method 2 (G);

VAL from water using method 1 (H) and method 2 (I).

tion was dependent on the source of VAL, solvents and

crystallization conditions

Dissolution studies

The dissolution profiles of VAL and its

recrystalliza-tion are shown in Fig 8 (China source) and Fig 9 (India

source) In general, the dissolution rates of crystals

obtained from solvents were slightly faster than for

the original VAL regardless of which method of sample

preparation was used However, it was notable that

there was a significant difference between samples

re-crystallized from acetone by method 1 and untreated

VAL (Fig 8A and Fig 8B) The samples obtained from

acetonitrile by method 2 also showed a significantly

faster dissolution rate compared with that of untreated

VAL (Fig 8B and Fig 9B) These results indicate that

the different recrystallization conditions and solvents

had a significant effect on the dissolution rate of VAL

The increase in dissolution rate could be explained by

the effect of the crystallinity of VAL (Adhiyaman and Basu, 2006; Youn et al., 2011) The reduction or disap-pearance of some peaks after recrystallization indicated that the crystalline structure of the drug changed into

a partially crystalline form, which attributed to the improved dissolution rate of VAL (Tran et al., 2009, 2010) In addition, the surface area of the various crys-tals with different shapes also affected the drug release rate (Nokhodchi et al., 2003; Uchimoto et al., 2011)

In conclusion, the crystalline modification of VAL from two different sources was investigated under various conditions The results of this study show that the polymorphic forms of VAL, confirmed by different analytical methods, were significantly affected by ex-perimental conditions during drug recrystallization DSC and PXRD show that there were differences in the structural behaviors of the drug after recrystalli-zation In addition, SEM also showed differences in the crystalline shape of the recrystallized samples

Fig 8 Dissolution profiles of pure VAL (China source) and

its recrystallized powders in water using method 1 (A) and

method 2 (B).

Fig 9 Dissolution profiles of pure VAL (India source) and

its recrystallized powders in water using method 1 (A) and method 2 (B).

Therefore,the dissolution rates of VAL were slightly

different among the polymorphic samples due to the

crystal rearrangement and morphology of VAL during

recrystallization The quality and physicochemical

properties of raw materials should be carefully

char-acterized for optimal dosage formulation

ACKNOWLEDGEMENTS

This work was supported by the Ministry of

Educa-tion, Science and Technology (BK21 Korea) and by a

grant from the Korean Health Technology R&D Project

(A092018), Ministry for Health, Welfare & Family

Affairs, Korea We would like to thank the Central

Research Laboratory for the use of the DSC, PXRD,

SEM and FTIR, Kangwon National University

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