Preparation of Norfloxacin Spherical Agglomerates Using the AmmoniaDiffusion System

HECTOR GABRIEL PUECHAGUT*, JORGE BIANCHOTTI, AND CARLOS A. CHIALE

Contribution from the Instituto Nacional de Medicamentos − A. N. M. A. T. − Av. Caseros 2161,1264 Buenos Aires, Argentina

Received November 14, 1996. Final revised manuscript received March 4, 1997. Accepted forpublication December 1, 1997.

Abstract 0 Agglomerated crystals of norfloxacin were prepared by aspherical crystallization technique using the ammonia diffusion system(ADS). This technique makes it possible to agglomerate amphotericdrugs like norfloxacin, which cannot be agglomerated by conventionalprocedures. When an ammonia−water solution of norfloxacin ispoured into an acetone dichloromethane mixture under agitation, asmall amount of ammonia is liberated in the system. The ammonia−water solution plays a role both as a good solvent for norfloxacin andas a bridging liquid, allowing the crystals’ collection to take place inone step. It has been proven that the agglomeration mechanismfollows three steps: first acetone enters into the droplets of ammonia−water (this emultion is formed because of the system characteristics);dissolved norfloxacin is consequently precipitated while the dropletscollect the crystals; simultaneously, a part of the ammonia containedin the agglomerates diffuses to the outer organic solvent phase, therebyforming the norfloxacin spherical agglomerates. The correct selectionof solvents has enabled us to obtain a suitable stable crystalline shape.

The quality and efficiency of a solid pharmaceuticalpreparation is influenced by primary micrometric proper-ties (shape, size of crystals, etc.) and macrometric proper-ties (bulk-density, flowability, etc.) from active and inactivemedical substances, especially when a large amount ofnonwater soluble drugs with poor reologic properties areformulated. The modification of these properties may beuseful to reach major active compounds with dissolution-improved bioavailability. Accordingly, an appropriatepharmaceutical design is desired.Kawashima and co-workers1,2 established an agglomera-

tion technique that transforms needlelike salicylic acidcrystals into a spherical shape during crystallization. Thistechnique, termed “spherical crystallization (SC)”, is usedto modify several properties of powder particles such ascrystalline form and particle size and shape (primaryfunction); flowability, packability, bulk-density, and ap-parent density (secondary function); pH, humidity, heat,and light (higher level multiple function).3 These modifica-tions allow for the practice of more efficient manufacturingmethods, which could save time and ultimately decreaseeconomic risks.The spherical crystallization, or particle spherical ag-

glomeration method, employs three solvents: one is thesubstance dissolution medium, another is a partially dis-solved medium for the substance, and the last one isimmiscible with the substance (e.g. water-ethanol-chloroform for acetylsalicylic acid). This process is carriedout controlling physical factors, such as agitation temper-ature, and chemical factors, such as solubility, rawmaterialconcentration, and solvent quantity.

Norfloxacin (Figure 1) is an antibacterial agent of thequinolone compounds, which is used to treat urinaryinfection. It has a zwiteronic molecular structure and thusis only soluble in acid or alkaline solutions. This is thereason a conventional technique to prepare sphericalagglomerates cannot be employed.Given that the reologic properties (micrometric and

macrometric) of this drug are not convenient, they weremodified to obtain a proper particle preparation using thespherical agglomeration technique combined with theammonia diffusion system (applicable to amphoteric drugs).

Materials and Methods

Chemicals and InstrumentssAnalytic grade solvents wereused (acetone and dichloromethane were from Merck and the* E-mail: gpuechag@anmat.gov.ar.

Figure 1sAnhydrous norfloxacin structural formula.

Figure 2sSpherical agglomeration equipment: (A) cylindrical vessel, (B)machine, (C) agitator turbine (four blade screw propellers), (D) double glassedwall (cooling jacket), (E) temperature regulator, (F and G) entrances, (H andH′) pipe line connections.

© 1998, American Chemical Society and S0022-3549(96)00463-7 CCC: $15.00 Journal of Pharmaceutical Sciences / 519American Pharmaceutical Association Vol. 87, No. 4, April 1998Published on Web 02/12/1998

ammonia hydroxide was from Carlo Erba), and the raw materialwas a product from Marshing & Co. Ltd. A/S, batch number 2039.The crystalline structure characterization was carried out using

the following equipment: ultraviolet-visible spectrophotometry,Shimadzu UV 2101PC; infrared spectrophotometry, Perkin-Elmer783, using KBr pellets and Nujol Liquid paraffin methods; meltingpoint, Buchi 510 semiautomatic fusiometer increasing 2 °C permin; optical, polarized light and hot stage microscopy, Carl ZeissAxioscop, with a Linkam TMHSG 600 hot stage; differentialscanning calorimetric characterization, Mettler TA 3000 andMettler DSC 20; thermogravimetric characterization, Mettler TG50 and Mettler M 75 Microbalance; and coulombimetric waterdetermination, Metrohm 625 KF.Solubility Measurement of Norfloxacin in Several Sol-

ventssThis measurement was made to find the highest norfloxa-cin concentration in a particular polarity range in order to choosethe right solvents for the working system. An excess amount ofnorfloxacin was added to 10 mL of various solvents such asacetone, dichloromethane, and ammonia-water solution 20% w/v,in a flask. The suspensions were shaken at 25 °C for 12 h.Samples were ultracentrifugated, aliquots of the supernatant wereevaporated, and the residue was diluted with 0.1 N hydrochloricacid. The norfloxacin contents in the medium were assayedspectrophotometrically at 274 nm. As a result, main solubilityvalues were obtained for dichloromethane (4.5 mg/mL), acetone(5.1 mg/mL), and the 20% (w/v) ammonia-water solution (15.3mg/mL).4

SC ProcesssAnhydrous norfloxacin was dissolved in enoughammonia-water (20% w/v) to 1.75% (w/v) and maintained at 40°C to avoid solubility problems. This solution (5.7 mL) was pouredinto a mixture of acetone (47.0 mL) and dichloromethane (13.0mL) under agitation at 150 rpm using a screw-type agitator in acylindrical vessel (200 mL) (Figure 2). The system was thermallycontrolled at 18 ( 1 °C throughout the process. After 5 min, thesolvent mixture (ammonia-water, acetone, and dichloromethane)

Figure 3sSpherical agglomeration mechanism using ADS: (I) invasion ofacetone into ammonia−water droplets, (II) diffusion of ammonia in theagglomerates to the outer solvent, (III) agglomeration ending.

Figure 4sTriangular phase diagram.

Figure 5sPolarized light microscopy of spherical agglomerates.

Figure 6sFlat rhomboidal crystalline view of crystal morphology.

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was removed, and the agglomerated crystals were washed withdichloromethane. Afterward, they were dried in a stove, at 50 °C,

for no more than 3 h, and were finally kept in a dark and dryplace.

Figure 7sOpened crucibles: (a) DSC of raw material and (b) DSC of spherical norfloxacin agglomerates.

Figure 8sClosed crucibles: (a) DSC of raw material and (b) DSC of spherical norfloxacin agglomerates.

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Results and Discussion

Selection of SolventssNorfloxacin is only soluble inacidic and alkaline solutions, reaching a maximum solubil-ity value of 12% (w/v) at pH 1.5 and of 11% (w/v) at pH10.5.4 To obtain norfloxacin agglomerates using the SCtechnique, a proper solvent was selected.5 Accordingly, a20% (w/v) ammonia-water solution was used because itspH ) 11. The other selected solvents were acetone (inwhich the drug is partially soluble) and dichloromethane(immiscible with water).Agglomerated Crystals and Constitutive Particless

When an ammonia-water solution of norfloxacin is pouredinto a mixture of acetone and a water immiscible solvent,such as dichloromethane, under agitation, an emultion isformed. After that, a small amount of ammonia diffusesout of the droplets to the organic solvent phase and itsability as a bridging liquid becomes weaker. Meanwhile,the acetone is added (solvent interchange) allowing nor-floxacin precipitation inside the droplets and determiningthe agglomerates’ size. Then the crystals’ nucleation beginsand the spherical agglomeration is induced. It is noticeablethat small crystals are needed to achieve good compactationas well as greater crystals surface. This process, originallydescribed by Ueda et al., is carried out at 18 °C5 (See Figure3).It is important to find a suitable combination among

solvents to reach the highest crystallization rate. The mostcapable region of agglomeration is shown by a shaded areain the triangular phase diagram for the ammonia-water/acetone/dichloromethane system (Figure 4). This is theregion where spherical agglomeration occurs. Above thisarea, the resultant agglomerates form a large solid mass

or a paste and below the broken line norfloxacin crystalsare dispersed without agglomeration.Crystalline Structure Characterization of Raw

Material (Anhydrous Norfloxacin) and SphericalAgglomeratessUsing the spherical agglomeration tech-nique with the ammonia diffusion system, a new pseudopoly-morph (trihydrate norfloxacin) was obtained. It wascharacterized by employing proper techniques, till theconfirmation that a new crystalline structure had beenobtained. The melting point was determined for anhydrousnorfloxacin and spherical agglomerates, which were softlydivided to avoid crystal trituration effects. Given that theraw material melting point range was 219.0-221.0 °C andthe agglomerated crystals’ melting point range was 219.5-221.5 °C, a crystal modification could not be confirmedsince the error in the method was ( 0.5 °C. After thesubstance melted, its color changed from light yellow todark red. This characteristic was recorded for furtherdeterminations. After that, microscopical studies to setmorphological differences between the treated and un-treated substances were carried out (e.g. optical, polarizedlight, and hot stage microscopy). The spherical agglomer-ates had a mean diameter of 400 µm, ranging from 100 to700 µm. Using optical and polarized light microscopy asuitable crystalline compactation was observed (Figure 5).Raw material crystals had a hexagonal-like crystallinestructure differing from agglomerated crystals, whichpresented a flat rhomboidal shape (Figure 6). Both crystalsshowed a birefringence phenomena. While using hot stagemicroscopy, no polymorphic change was observed. Colora-tion appeared after reaching the melting point, which couldbe due to substance decomposition. By increasing theagglomerated crystals’ temperature to 104 °C in a siliconebath, a substance was vaporized (it might have beencrystallization water). Considering these data, the ag-glomerated crystals could present a pseudopolymorphicstructure. This reticule modification was further charac-terized by DSC, TG, and IR.Open-crucible and closed-crucible DSC methodologies

were performed for both materials. The first one wasperformed to observe volatile substances lost (nitrogen flowwas not considered necessary). The agglomerated crystals’sample weight was recorded before and after the determi-nation, indicating a sample component loss at 104 °C withan endothermic event (434.73 J/g) (Figure 7b). This couldbe a water loss presenting a stoichiometric ratio of water/norfloxacin (3/1). The presence of water was confirmedcoulombimetrically, and a trihydrate norfloxacin moleculewas established. Closed methodology allowed us to observethat water molecules were gradually liberated, which isconsistent with its place in the crystalline structure.Within the range of 95-210 °C three endothermal peaksappear at 140, 180, and 190 °C (peaks 1, 2, and 3),indicating a gradual liberation of water, with each endo-thermal peak representing the loss of one water moleculefrom the crystalline reticule (Figure 8b). The melting pointthat was recorded using this technique was 218.8 °C (peak4). The same procedure was used with raw material,determining a melting point of 218.2 °C, while no thermalevents were previously observed (Figure 8a). Comparativecharts between agglomerates and raw material determina-tions appear in Figures 7a and 8a, the raw materialthermogram being considered as a control. To employ theprevious methodology, the agglomerates needed to bebroken to avoid comminution trituration effects. Thistreatment did not generate any modification or polymorphicinstability to the raw material or to the norfloxacinagglomerates. The thermogravimetric characterizationwas consistent with DSC results, confirming the thermalnature of trihydrate norfloxacin crystals. The water loss

Figure 9sThermogravimetric characterization of spherical agglomerates.

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(107.3 °C) corresponded to a 15% weight decrease (3/1water norfloxacin ratio) (Figure 9).Given that the humidity content was 0.91%, our raw

material complied with the USP 23 specifications.Infrared spectrophotometry allowed us to certainly char-

acterize the water state inside the crystals of the sphericalagglomerates and its chemical interaction with the nor-floxacin molecules. The spectrums of the substances(norfloxacin agglomerates and raw material) using KBrpellets and Nujol liquid paraffin methods did not presentgreat fingerprint differences. Comparing both materials,changes such as CdO stretching were observed, from 1725cm-1 for raw material to 1635 cm-1 for spherical agglomer-ated crystals, possibly due to the formation of intermolecu-lar hydrogen bond. The greater amount of water contentin the agglomerated crystals is also evident.

ConclusionsAccording to these results, we may establish that the

molecular modification, with a pseudolpolimorphic nature,appears in the constitutive crystals of the agglomerateswhen compared with the raw material.Preparation of particles using spherical agglomeration

has become an important subject. It optimizes medicineproduction, by reducing costs, employing simpler method-ologies (such as direct compression for tablets and micro-

spheres envelopes), and improving the active compounds’bioavailability whenever it is possible. Using the sphericalcrystallization technique combined with the ammoniadiffusion system a new pseudopolymorph (trihydrate nor-floxacin) was obtained. This enabled us to achieve bettermicrometric and macrometric properties. Future studieswill determine the pharmacotechnical properties of nor-floxacin tablets and study the availability of this newcrystalline structure.

Acknowledgments

To Dr. L. L. A. Pineyro de Castellano, Dr. J. M. E. Ciura and toMiss K. Bok for their help and technical assistance.

References and Notes1. Kawashima, Y.; Okumura, M.; Takenaka, Y. Science 1982,

216, 1127.2. Kawashima, Y.; Okumura, M.; Takenaka, Y.; Kojima, A.

Pharm. Sci., 1984, 73, 1535.3. Kawashima, Y. Kona 1987, 5, 69-75.4. Mazuel, C. Anal. Profiles Drugs Substances 1991, 20, 557-

600.5. Ueda, M.; Kawashima, Y. Chem. Pharm. Bull. 1990, 38 (9)

2537-2541.

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