J Therm Anal Calorim DOI 10.1007/s10973-014-3691-9

Preformulation study of ivermectin raw material Larissa Arau´jo Rolim • Fla´via Ca´ssia Maria dos Santos • Luı´se Lopes Chaves Maria Luı´za Carneiro Moura Gonc¸alves • Jose´ Lourenc¸o Freitas-Neto • Andre´ Luiz da Silva do Nascimento • Jose´ Lamartine Soares-Sobrinho • Miracy Muniz de Albuquerque • Maria do Carmo Alves de Lima • Pedro Jose´ Rolim-Neto

•

Received: 30 January 2013 / Accepted: 9 February 2014 Ó Akade´miai Kiado´, Budapest, Hungary 2014

Abstract The aim of this study was to characterize the raw material ivermectin (IVC) using different analytical techniques used for drugs with its pharmacological characteristics. Mass spectroscopic and infrared absorption analyses were carried out to identify the molecule, and further analyses to confirm its crystalline structure (electron sweep microscopy and X-ray diffraction), as well as granulometric analysis, apparent, and compacted density, leading to the conclusion that, even with a crystalline structure IVC has good flow and compressibility. Differential Scanning Calorimetry and thermogravimetric analysis were used for the infrared thermal characterization, determination of the melting point (157 °C), initial degradation temperature (305 °C), loss of mass with the increase of the temperature (3 events, the first dissolution and degradation in two consecutive stages). Using the aforementioned techniques, it was possible to carry out a compatibility study of IVC with some excipients used in solid pharmaceutical form, which demonstrated an incompatibility between IVC and lactose and amide. These results can be used to develop new pharmaceutical forms and for more rational quality control of forms already commercialized, with better understanding of the characteristics of IVC. Keywords Physico-chemical characterization
Raw material
Analytical techniques L. A. Rolim
F. C. M. dos Santos
L. L. Chaves
M. L. C. M. Gonc¸alves
J. L. Freitas-Neto
A. L. da Silva do Nascimento
J. L. Soares-Sobrinho (&)
M. M. de Albuquerque
M. do Carmo Alves de Lima
P. J. Rolim-Neto Universidade Federal de Pernambuco, Av. Prof. Arthur de Sa´ S/N, Recife, PE, Brazil e-mail: [email protected]

Introduction Ivermectin is a semi-synthetic product obtained from avermectin, naturally synthesized by the microorganism Streptomyces avermitilis. It consists of a mixture of two homologs dihydroavermectin B1a (H2B1a) and dihydroavermectin B1b (H2B1b) [1]. It is used as an active ingredient with broad-ranging medical applications for the treatment of rashes, worms, and lice, acting on the nervous system and functioning of the muscles, resulting in paralysis and death of the parasites [2]. Ivermectin should be considered as a critical drug, whose physical and chemical characteristics need to be controlled in the pharmaceutical industry. Chemically, IVC is a mixture of structural isomers, as mentioned, which act in different ways and have different levels of toxicity. It being expected that the raw material does not have less than 80 % of the B1a isomer [3], while physically it presents itself as a poorly soluble drug with crystalline structure (class II in the biopharmaceutical classification). All these critical points need to be controlled when obtaining technological forms of the drug. The concept of quality by design (QbD) is systematic and scientific, based on integral risk, and a proactive approach to the development of medicines. It starts out with previously defined objectives and emphasizes the product and understanding of the manufacture process and the control of processes [4]. QbD means guaranteed quality, improving the scientific methods that should be used in the research and development and design phases, so that the processing of the product is as quick as possible [5, 6]. QbD identifies characteristics of drugs that are critical for quality from the point of view of production, which translates into attributes that the drug should have to insure safety and efficiency for users.

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Thus, there is an imminent need to review and refine the physico-chemical characterization of IVC, since the pharmacopeia tests required at present are not sufficient to identify subtle differences between raw materials. This evaluation may be one of the tools for establishing the quality of the raw material, which, even though it originates from various manufacturers and is used for the manufacture of pills, should provide discrimination of results and consistent interpretations.

Experimental Materials Two batches of ivermectin were used, one of raw material provided by Laborato´rio Veterina´rio Valle´eÒ (Batch 07016/2010, made in China), and the standard ivermectin acquired from Sigma AldrichÒ, batch 70288-86-7 (purity: 95 % H1B1a ? 2 % H1B1b) to calc the purity of raw material used. The binary mixtures (BM) 1:1 (p/p) of IVC with excipients were prepared using a mortar and a pestle, breaking down the mixtures for 3 min each. The BM were prepared using the excipients: microcrystalline cellulose (MCC), talc, polyvinylpyrrholidone (PVP) K-30, sodium croscarmellose, StarchÒ (pre-gelatinized starch), StarlacÒ (85 % monohydrated lactose ? 15 % starch—spray-dried compound), FlowlacÒ (spray-dried monohydrated lactose), TabletoseÒ (monohydrated lactose), and AerosilÒ (colloidal silicon dioxide).

Methods Chemical identification The infrared with fourier transform (FT-IR) spectrum were obtained using a Spectrum 400 PerkinElmerÒ with an attenuated total reflectance device (ATR) with a selenium crystal. The samples to be analyzed were transferred directly to the compartment of the ATR device, and the result obtained using the mean of 10 sweeps, from 650 to 4000 cm-1 at a resolution of 4 cm-1. In addition to IR identification, mass spectrometry was also carried out using a ShimadzuÒ IT-TOF machine, with ionization by thermal nebulization and flight time mass analyzer, the drug being diluted using an acetonitrile:water system (50:50), by way of positive ionization, with a 80–950 m/z scan. Content determination and solubility study The samples and the working standard IVC were obtained using an initial dilution of 10 mg of IVC in solution of

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acetonitrile:methanol:water (26:64:10), with 5 min of stirring by sonication, in a 100 mL volumetric flask, to obtain a final concentration of 200 lg mL-1, with subsequent dilutions to 50, 100, and 150 lg mL-1 to obtain the calibration curve for the raw material and plot three authentic curves for 50, 75, 100, 125 and 150 lg mL-1 to determine the linearity of the IVC standard, making it possible to determine the IVC content of the raw material used for this study. First, the solutions obtained were submitted separately to HPLC–DAD analysis, initially following the parameters laid out by Cione and Silva [7], which involve the use of a C-18, 250 9 4.6 mm (5 lm) column, 25 °C, as the stationary phase, a 0.2 % methanol:acetonitrile:acetic acid solution (260:640:100 mL v/v), as the mobile phase, with a flow of 1.5 mL min-1 at 254 nm. After a number of analyses had been conducted, the method was optimized, modifying the composition and the flow of the mobile phase to 0.2 % methanol:acetonitrile:phosphoric acid (260:640:100 mL v/v), with a flow of 1 mL min-1 at 254 nm with completed validation of this new method according ICH Q2(R1). The solubility of IVC was determined by two studies. An initial semi-quantitative study [3, 8] is to test the solvents: ethyl acetate, acetone, acetonitrile, 0.2 M hydrochloric acid, water, absolute ethyl alcohol, dichloromethane methyl alcohol, ethylic ether, 0.2 M sodium hydroxide, n-hexane, and 3 % hydrogen peroxide [5, 8]. In parallel, A quantitative study was also conducted using the method described for determining the IVC content by way of HPLC–DAD, in which IVC samples were added to solutions of: water, pH 6.8 (50 mL of H2KPO4 0.1 M solution ? 23.65 mL NaOH 0.1 M solution ? water q.s.p 100 mL), pH 4.0 (9.35 mL 0.2 M Na2HPO4 ? 10.65 mL 0.1 M citric acid ? water q.s.p. 100 mL), pH 1.2 (50 mL buffer solution H3BO3
KCl 0.1 M ? 97 mL 0.2 M HCl solution ? water q.s.p. 200 mL) with and without 1 % sodium lauryl sulfate (SLS), until a saturated solution was obtained (with aliquots removed at 24, 72, and 168 h). Determining the physical and particle characteristics The diffractograms for the drug were obtained using a SIEMENSÒ diffractometer (X-Ray Diffractometer, D-5000), equipped with a copper anode. The samples were analyzed at the 2h angle interval of 2–60 at a digitalization speed of 0.02° 2h s-1. The samples were prepared on glass support with a fine layer of powder material without solvent. The crystal morphology of IVC was examined using an electron sweep microscope JeolÒ JSM-5900, after being fixed on double-sided carbon tape and metalized with gold for 15 min (Metalizer BaltecÒ SCD 050). The electromicrographs were obtained using a camera with an excitation tension of 15 kV.

Preformulation study of ivermectin raw material

The distribution of particle sizes was determined by sieving, using standardized stacked sieves (0, 75, 90, 150, 250, 425, 500 lm), mounted on a base equipped with magnetic vibration (Tamizador BertelÒ) for 20 min, in triplicate. The density of the powder was determined by an assay with 10 g of the samples in an automatic compactor (Tap Density, VarianÒ) equipped with a standard test tube, in triplicate [7]. The initial volume occupied by the product was measured, and then 10 compactions were carried out to accommodate the powder. Then, further 1,000 consecutive compactions were carried out until no change was observed in the volume. The relation between the mass of the samples and the volume occupied by the powder before and after compaction determined the apparent density (dAP) and the compacted density (dCP). The compaction capacity of the powder (10 g) was evaluated using the Hausner Index (HI) and the Carr Index (CI) using the following equations: HI = dCP/dAP e CI (%) = (dCP - dAP)/ dCP 9 100, respectively. The angle of repose was measured by the cone of powder formed by running the drug through a funnel of standard dimensions onto a flat surface, with 10 g of IVC. The flowing time was determined by the average time needed for a pre-established quantity of drug to run through the funnel, using a digital stop watch.

Thermal characterization of IVC The thermal characterization of IVC was carried out using differential scanning calorimetry(DSC) and thermogravimetric analysis (TG). The DSC curves were obtained using a ShimadzuÒ DSC-60 calorimeter connected to ShimadzuÒ TA-60WS software in a nitrogen atmosphere of 50 mL min-1 at various heating rates (5, 10, 15, and 20 °C min-1) in the temperature range of 25–300 °C. The samples were placed in a hermetically sealed aluminum sample holder with masses of 2 mg (±0.2) of sample, in triplicate. Indium and zinc were used to calibrate the equipment in terms of the temperature scale and enthalpy response. The TG analyses were carried out using a ShimadzuÒ, TGA Q60 thermoscale, in a nitrogen atmosphere flowing at 50 mL min-1, with a sample mass of around 5 mg (±0.4), processed in a platinum bowl for the temperature range of 25–600 °C at a heating rate of 10 °C min-1. Prior to the assays, the thermoscale was checked using hydrated calcium oxalate. The kinetic investigation of the non-isothermal degradation of IVC was obtained using TG data collected by applying the Ozawa method. The heating rates used were 2.5, 5.0, 10, 20, and 40 °C min-1, within a temperature range of 30–600 °C, in platinum bowls with approximately 5 mg of sample in a dynamic N2 atmosphere (50 mL min-1).

Binary Mixtures

DSC / TG No Interaction Signals?

Compatible

Yes

Degradation Studies Ozawa

FT-IR-ATR

Fig. 1 Flowchart of study of drug-excipient compatibility

Study of drug-excipient compatibility The fluxogram presented in Fig. 1 shows the protocol used to carry out the study. In the first stage, IVC analyses were carried out separately and in BM by DSC and TG, with values referring to the initial melting point (Ti) and the initial temperature of decomposition (Td) adopted as indicative of the reaction between the drug and the excipient. The TG analyses used to calculate the degradation kinetics, and FT-IR followed the same parameters described for the analyses of IVC isolated.

Results and discussion Chemical identification of IVC Absorption spectrometry in the infrared region (IR) is nowadays one of the main resources for structural identification of organic substances, and it is of extreme importance in the control of the quality of raw materials and excipients, as comparing spectra obtained from the raw material with reference substances may reveal early degradation, or characterize technological processes as in the case of micro- and nano-particles which should normally bond weakly with drugs to help improve their solubility or vectorization. These bonds can be seen in the FT-IR spectra. The two isomers present in IVC raw material are differentiated by a CH3 group in the structure as illustrated in Figs. 2 and 3. Organic compounds with ether groups normally display characteristic bands related to C–O bonds, as is the case of the IVC molecule which is composed only of

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L. A. Rolim et al. Fig. 2 Chemical structure of IVC

H3C

H3C

CH3

O O

HO

CH3 H

O

O O

H3CO

R

H3CO H3C O

CH3

Bla

OH

CH3

R= CH3

O

O CH3

Blb

897,48 100 Positive ionization 80

%

60 478,27

40 20

913,47

284,30

0 200

400

600

800

1000

1200

m/z

Fig. 3 Mass spectrum of IVC

atoms of carbon, oxygen, and hydrogen, forming rings and hydroxyl groups, ethers and a single ester group. In the spectra obtained (Fig. 8), it was possible to identify 6 regions as inherent characteristics of IVC: at 3,500 cm-1, relating to axial deformation of O–H; 2,964.99 and 2,937.33 cm-1, characteristics of methyl groups, axial deformation of C–H; 1,728.92 cm-1, characteristic of saturated aliphatic ketone; 1,675.79 cm-1 indicating unsaturated lactones with a double bond adjacent to the –O– group, owing to the C=C group; between 1,383.87 and 1,313.84 cm-1 showing moderate absorption of ketones, in consequence of the axial and angular vibrations; 1,198.96 and 1,182.48 cm-1 indicating absorption of esters by lactones; between 1,142.13 and 1,021.91 cm-1 shows the absorption more characteristic of aliphatic ethers, owing to the asymmetric axial deformation of C–O–C; 982.11 and 970.57 cm-1 and the two symmetrical angular deformation bands outside the =C–H plane of the terminal alkenes; 950.78 and 929.74 cm-1 show angular deformation outside the O–H plane; 904.25–832.48 cm-1 these intense bands in

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R=

CH3

H OH

the low-frequency region derived from angular deformation outside the plane of the C–H bonds of the ring; 807.96–706.11 cm-1 indicate angular deformation outside the C–H plane; and 686.98 and 661.13 cm-1 angular deformation outside the C=C plane of the rings. The mass spectrum of IVC is very peculiar, thus it was not possible to detect the raw mass of the drug as a characteristic peak, the strongest peak was found at 897 m/z, which corresponds to an H2B1a ? Na? dimer, followed by secondary peaks at 284, 478 m/z, related to the fragmentation of ionization, and 913 m/z (H2B1a ? K?). If IVC did not have this capacity to form complexes the mass spectrum should have two peaks with different intensities according to the quality of the raw material: 874 m/z (CH2CH3–H2B1a) and 860 m/z (CH3–H2B1b) [9]. Content determination and solubility study (HPLCDAD) IVC is obtained from the biosynthesis of microorganisms such as S. avermectinius, which synthesize not only ivermectin but also other avermectins, and the pharmaceutical raw material thus passes through a series of purification/ recrystallization steps in which the specification of purity to be used as the pharmaceutical raw material is not less than 80 % of H2B1a and no more than 20 % of H2B1b calculated for an anhydrous sample, free of ethanol, and dimethylformamide [10, 11]. The result of the average content study using HPLC– DAD for the raw material was 93 ± 0.9 % of H2B1a in relation to the standard, which is suitable for use, according to official compendia [3, 12]. The absorption of drugs in solid orally administered pharmaceutical forms depends on their release and absorption under physiological conditions and the permeability of the biological membranes. Based on these considerations, it can be said that the dissolution of the drug in

Preformulation study of ivermectin raw material 6000

Table 1 Result of semi-quantitative solubility study of IVC Solubilization volume/mL

Classification

Descriptive term

5000

Ethyl acetate

10

Easily soluble

From 1 to 10 parts

4000

Acetonitrile

30

Soluble

From 10 to 30 parts

Acetone

100–1,000

Poorly soluble

From 100 to 1,000 parts

Water

[10,000

Practically insoluble

[10,000 parts

Ethyl alcohol

30–70

Slightly soluble

From 30 to 100 parts

Methyl alcohol

30

Easily soluble

From 10 to 30 parts

Dichloromethane

10

Practically insoluble

From 1 to 10 parts

Ethylic ether

30

Practically insoluble

From 10 to 30 parts

H2O2 3 %

[10,000

Practically insoluble

[10,000 parts

HCl 0.2 M

[10,000

Very poorly soluble

[10,000 parts

NaOH 0.2 M

[10,000

[10,000 parts

N-Hexane

1,000–10,000

From 1,000 to 10,000 parts

Table 2 Quali-quantitative determination of solubility in aqueous systems Samples

Time/h

Content (lg mL-1)

H2O

168

–

H2O ? SLS 1 % pH 1.2

72 72

Intensity/%

Solvent

9,32

3000

2000

13,16

18,12 18,7

1000

0 5

10

15

20

25

30

35

40

45

50

2θ /° Fig. 4 Diffractogram and electromicrograph of IVC crystalline particles

4 lg mL-1 [13]. The other samples were analyzed at 72 h, since the media used were already saturated in this time. In addition, regarding the results obtained from the solutions with different pH, it was observed that the solubility increases with the increasing of the pH of the solution. This result was expected once the molecule is extremely apolar, with a pKa value around 6.5, what means that the molecule must be in a solution with a very alkaline pH to be ionized, what may lead to the increase of the solubility. These results were important to predict the solubility and consequently the permeation of IVC in biological fluids, which can help to understand the absorption profile, and thus the effectiveness.

10.43 819.41

Determination of physical and particle characteristics X-ray diffraction is a highly versatile and rapid technique for the application of polycrystalline samples, such as monitoring samples in the development of pharmaceutical products in the laboratory and industrial quality control, providing information on the size and structure of crystals. This technique is based on the principal that when a material is exposed to monochromatic X-rays, for a perfectly aligned crystal, in which the atoms are regularly packed and the distance between the crystallographic planes is defined by the physical characteristics of the sample, which can be used precisely to measure the spaces in the crystalline reticle. This assay established the diffraction pattern of the IVC powder, revealing the presence of a series of peaks, with a distinct peak at 2h around 9.32°, apart from other secondary lower intensity peaks at 9.04°, 12.36°, 13.16°, 13.56°, 18.12°, 18.7°, and 27.32° (Fig. 4), showing the typically crystalline behavior of the drug, which may be relevant to its solubilization and fluidity.

pH 1.2 ? SLS 1 %

72

61.86

pH 4.0

72

11,403.30

pH 4.0 ? SLS 1 %

72

259.01

pH 6.8

72

21,747.73

pH 6.8 ? SLS 1 %

72

23,756.06

an aqueous medium is a limiting stage. The result of the solubility study for IVC revealed low solubility in water (Table 1), confirming the results already reported in the literature [3]. For a more detailed evaluation a quantitative study of solubility in aqueous systems was carried out using HPLC– DAD, the results of which are described in Table 2. It can be inferred from the results obtained that the aqueous solubility of IVC is below the quantification limit of the method (QL = 6.58 lg mL-1), calculated using the standard linearity curve (y = 3568.51x - 30518.48), which corroborates the finding in the literature of

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L. A. Rolim et al.

at 2 ± 1 s, when tested a mass of 10 g. This behavior diverges from that expected given the crystalline morphology of the particles (Fig. 6), which favors interaction between the particles. The drug can therefore be processed by way of direct compression, since, despite the crystalline structure of IVC, it has a good flow and good compressibility.

30 25

%

20 15 10

Thermal characterization of IVC

5 0 0

100

200

μm

400

500

Fig. 5 Distribution of frequency of IVC particle size

Table 3 Results for compaction properties of sample under analysis Parameters

Values obtained

-1

dap/g mL

0.67

dcp/g mL-1

0.74

IH

1.10

CI

9.46

The granulometric distribution carried out (Fig. 5) showed that the particles of the raw material used for the study tend to cluster in the 90- to 250-lm interval. In the light of these results, an evaluation was carried out of the retention in relation to the passage of particles in the intervals under study. It was shown that the mean size of the particles is approximately 198 lm. The raw material used can thus be classified as a semi-fine powder [6]. This knowledge is of great importance, given that the speed of dissolution is directly proportional to the surface area of particles and, as dissolution is a critical factor for this drug, determining the size and morphology of IVC particles allows these factors to be correlated. The physical and mechanical properties of the compaction and rheology of IVC were determined, and the results are shown in Table 3. The data showed that the density of IVC was accordingly with those reported in the literature. However, the Carr Index expressed in the form of a percentage the compaction capacity of the powder under analysis, where values up to 10 % are considered to demonstrate excellent flow and compaction, as observed in the case of IVC. Furthermore, the Hausner Index, which is similar to the CI, lies between 1.00 and 1.11 demonstrating that the flow is excellent and there is no need for the addition of lubricants to improve the flow, while higher values demonstrate poor flow. Likewise, by determining the angle of rest, it was observed that IVC flows freely (angle of rest = 28° ± 3°),

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The thermo-analytic methods outlined in the fifth edition of the Brazilian Pharmacopeia of 2010 have been widely applied to the study of drugs. Many studies have used these methods as alternatives for the characterization and quality control of pharmaceutical materials. The DSC curves obtained to confirm the melting range of IVC (Fig. 6) at heating rates of 5, 10, 15, and 20 °C min-1 showed an endothermic peak in the 152.96–164.2 °C temperature range (mean), characteristic of the melting of the drug, although the melting peak was different when analyzed at different rates, occurring sooner the smaller the heating rate used. According to the Merck Index, 1999 [13], the melting band of IVC is 155–157 °C, and the most appropriate heating rate for thermal evaluation of IVC is 10 °C min-1 which allows a melting point of 157.4 ± 0.7 °C to be determined. The purity of IVC was also calculated using Van’t Hoff equation in the linearization of the melting event with analysis obtained at a rate of 0.5 °C min-1, confirmed in triplicate (and HPLC–DAD analysis, as described above). In this model, purity is determined through the deviation from linearity of the melting point, which occurs because of the presence of impurities. Knowledge of the deviation from linearity allows the correction factor in linearization of the straight line to be inferred. The purity of IVC was found to be around 98.4 %, with a calculated correction factor for impurities of 8.36 %, which does not concur with the content found using HPLC–DAD, as this technique is inappropriate for determining the purity of IVC, probably because of the enthalpy involved in the process being related not only to the melting of the drug, but also to the evaporation of residual solvents as will be discussed below. The TG curves provided information on the composition and thermal stability of IVC, thereby making it possible to determine the initial decomposition temperature for the raw material and the stages in the degradation of IVC, tracking any possible dehydration, oxidation, combustion or decomposition reactions. The TG curves for the raw material and the analytical pattern showed that IVC has three characteristic thermal events, the first loss of mass occurs when the drug melts, between 153 and 164 °C (Dm = 5.04 %), a second stage of decomposition between 312 and 327 °C (Dm = 58.54 %),

Preformulation study of ivermectin raw material Fig. 6 DSC curves for establishing heating rate for IVC

DSC mW 154.52C

0.0

5 °C/min 157.23C 10 °C/min

–10.0

158.29C 15 °C/min 160.03C

Endo

20 °C/min

–20.0 50.0

100.0

150.0

200.0

250.0

300.0

350.0

400.0

Temperature/°C

40.0

TG sample 2 Endo

TG sample 1

80

20.0 DTA sample 1

60

0.0 DTA sample 2

40

–20.0

20

Heat flow/mW mg–1

100

Mass/%

Fig. 7 TG curves for IVC without pre-heating (sample 1) and with pre-heating to 157 °C (sample 2) in an atmosphere of N2 at 50 mL min-1

–40.0 –0 –0

50

100

150

200

250

300

350

400

450

500

550

600

Temperature/°C

100 90 80

T/%

followed by a third and last degradation stage between 341 and 427 °C (Dm = 29.35 %) related to the carbonization of IVC (Fig. 7), events determined by the first TG derivative. The IVC raw material has formamide and ethanol in its crystalline network, which are solvents used for purification/recrystallization of the mixture of avermectins produced by biosynthesis of microorganisms. According to the specifications contained in the official compendia, analysis of these residual solvents should be carried out by way of gaseous chromatography (GC) with a maximum of 5 % ethanol and 3 % dimethylformamide [3]. However, it was found that TG analysis is also capable of quantifying the total residual solvent content, corresponding mostly to the first event involving loss of mass the IVC raw material undergoes. To prove that the first event involving loss of mass in IVC is not properly related to degradation, but a desolvation of ethanol and dimethylformamide, one TG analysis was carried out on a sample without pre-heating (sample 1) and another with an IVC sample pre-heated to the melting point, with subsequent re-cooling and re-heating of the sample to 600 °C (sample 2), curves shown in Fig. 7. The analysis of the TG curves obtained shows that, on the first heating, the solvents evaporate (Tboiling ethanol = 78.4 °C and Tboiling dimethylformamide = 153 °C) [14], which may justify the decreasing in the melting point

70 60 Sample 1

50

Sample 2 40 30 4000

3500

3000

2500

2000

1500

1000

cm–1 Fig. 8 FT-IR spectrum of samples of IVC without pre-heating (sample 1) and with pre-heating to 157 °C (sample 2)

of IVC (157 °C) in the second heating, once this temperature, coincides, approximately, with the boiling point of the dimethylformamide, corresponding to 6.5 % on average, a value similar to that calculated by CG for the analytical standard (sum of the data provided by Sigma AldrichÒ relating to the quantity of dimethylformamide and ethanol in the batch used in this study) added to the water content by Karl Fisher.

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L. A. Rolim et al.

(a) 100 Rz 40 °C min–1 Rz 2,5 °C min–1

Mass loss/%

Fig. 9 TG curves and Ozawa graph for IVC obtained for five heating rates under a dynamic nitrogen atmosphere using a non-isothermic method

70 Rz 5 °C min–1 50

Rz 20 °C min–1

Rz 10 °C min–1

20

0.0 100

200

300

400

500

Temperature/°C

(b)

LOG A

2.00

1.50

1.00

0.50

0.00

1.42

1.62

1.52

× 10–3

1/T/K

(c) 10.00

8.00

217.47 KJ mol–1 Kinetic Energy Order 2.0 Frequency Factor 1.539 × 1016 min–1

6.00

4.00

0.00

1.00

2.00

3.00

4.00

× 10–18

Reduced time/min

FT-IR analysis was also carried out on a sample preheated until the melting point of IVC (sample 1) and a sample without pre-heating (sample 2) as shown in Fig. 8, revealing the permanence of the characteristic bands of IVC even after heating, with no signs of degradation of the drug. TG analysis of IVC is thus characterized as an auxiliary technique for determining the quantity of residual solvents resulting from purification/recrystallization of this drug.

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As the first event involving loss of mass on the TG curve does not constitute a decomposition event, non-isothermic kinetic analysis of IVC was carried out to evaluate only the second mass loss event (310–330 °C) [14, 15]. In these analyses the TG curves shift to higher temperatures as the heating rate increases (2.5, 5, 10, 20, and 40 °C min-1), allowing the application of the Ozawa method, through good correlation between the five heating

Preformulation study of ivermectin raw material Table 4 Thermoanalytical data for IVC and BM with excipients Samples

Tonset/°C

Tmelting/°C

DH/J g-1

Tonset degradation/°C

% Degradation

IVC

144.04

159.42

-134.58

305.29

-53.59

IVC ? PVP K-30

149.54

160.01

-36.24

307.49

-27.62

IVC ? Talc

144.12

157.8

307.06

-50.02

-152.8

IVC ? CMC

147.18

159.53

-33.18

310.12

-34.59

IVC ? StarchÒ

145.47

161.28

-33.92

297.83

-63.88

IVC ? TabletoseÒ

142.9

149.9

-129.33

292.77

-59.6

IVC ? Croscarm.

144.57

156.57

-26.37

305.69

-51.92

IVC ? FlowlacÒ

141.27

149.35

-137.44

284.99

-50.74

IVC ? StarlacÒ

144.98

150.91

-99.4

293.66

-60.69

IVC ? Glycolate

144.06

156.5

-35.45

306.3

-52.4

IVC ? AerosilÒ

147.9

162.63

-59.58

308.36

-27.36

Table 5 Activation energy (Ae), frequency factor (A) of BM obtained by non-isothermic kinetics (Ozawa method) Sample

Activation energy (Ae)

Frequency factor (A)

IVC

216.03 kJ mol-1

1.620 9 1016 min-1

180.21 kJ mol-1

2.985 9 1013 min-1

-1

7.632 9 1014 min-1

IVC/TabletoseÒ IVC/Starch

Ò

200.44 kJ mol

IVC/TABLETOSE

rates. For this thermal decomposition, the Ae was calculated to be 217.47 kJ mol-1, with a second-order degradation reaction and a factor of frequency of collisions between IVC molecules of 1,539 9 1,016 min-1 (Fig. 9).

TABLETOSE

Drug-excipient compatibility study The behavior of binary mixtures showed that, with all excipients, there was a significant decrease in the enthalpy involved in the melting process, when compared with the melting enthalpy of the drug alone, as well as small changes in the shape of the peak, with few variations in the melting temperature, suggesting no incompatibility in most cases. The data from the TG and DSC curves obtained in the compatibility study are shown in Tables 4 and 5. In the DSC curves of the BM of IVC with lactose excipients (TabletoseÒ and FlowlacÒ), there was a reduction in the melting temperature of 1–3 °C from the start of the melting temperature, which may indicate the occurrence of a drug-excipient interaction. Furthermore, the initial degradation temperature (the second event involving loss of mass) decreased by 10 °C in the BM with lactose and amide (StarchÒ, TabletoseÒ, FlowlacÒ, and StarlacÒ), according to the TG/DTG curves. TabletoseÒ was therefore selected to represent excipients containing lactose and StarchÒ for those containing amide and the non-isothermic kinetics was evaluated at rates of 10, 20, and 40 °C min-1 for the IVC raw material and the BM containing these excipients.

IVC/Starch

STARCH

IVC

4000 3500 3000 2500 2000 1500 1000

cm–1

Fig. 10 FT-IR spectrum of IVC, TabletoseÒ, StarchÒ, IVC/TabletoseÒ binary mixture, IVC/StarchÒ binary mixture

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L. A. Rolim et al.

The activation energy of the IVC/TabletoseÒ binary mixture decreased by more than 15 % compared with IVC alone, while the IVC/StarchÒ mixture presented a reduction of 7 % in the activation energy, corroborating the results of the thermal analysis, which produced evidence of interaction. The FT-IRIV absorption spectra for IVC and the BM with the selected excipients are shown in Fig. 10. In all the spectra, there is only superposition of the characteristic bands of IVC in isolation and of the excipients, some of these having bands relating to the excipients superposed on them, although it should not be considered incompatibility, and the FT-IR technique was thus not selected to provide evidence of possible incompatibilities between IVC and the excipients under evaluation.

Conclusions Physico-chemical characterization confirmed data reported in the scientific literature, insuring the authenticity of the material under analysis. Quantification of the purity of the drug showed a good correlation between the data obtained using chromatographic and thermo-analytic techniques, showing that thermoanalytical techniques can be a powerful tool for evaluation of the purity of the substance. The Ozawa method showed the second-order kinetic behavior for decomposition of the drug, and the frequency factor for collisions between IVC molecules of 1,539 9 1016 min-1. The compatibility studies using DTG and TG showed that the drug-excipient association between IVC, TabletoseÒ and StarchÒ produces an increase in the percentage decomposition, as well as decrease in the Ae, seen in the analyses of non-isometric kinetics. The results obtained are thus of fundamental importance, as they have enabled the determination of the principle physical and chemical properties of IVC, providing relevant information on the quality of raw material used and its behavior under a range of different techniques frequently used to characterize pharmaceutical products, which have been being developed for this drug as a way of overcoming this characteristic which represents a technological and therapeutic barrier in the case of IVC, an antihelminth drug used to combat filariasis.

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