Article
`Solid-State Characterization of Different Crystalline
`Forms of Sitagliptin
`
`, Laiane J. Oliveira 1, Elisa F. Montin 1,
`Nayana C. F. Stofella 1, Andressa Veiga 1
`Itamar F. Andreazza 1, Marco A. S. Carvalho Filho 2, Larissa S. Bernardi 3, Paulo R. Oliveira 3
`and Fábio S. Murakami 1,*
`1 Departamento de Farmácia, Universidade Federal do Paraná, Av. Pref. Lothário Meissner, 632-Jardim
`Botânico, Curitiba 80210-170, Paraná, Brazil
`Escola de Ciências da Saúde, Universidade Positivo—UP, R. Prof. Pedro Viriato Parigot de Souza, 5300,
`Curitiba 81280-330, Paraná, Brazil
`Programa de Pós-Graduação em Ciências Farmacêuticas, Universidade Estadual do
`Centro-Oeste (UNICENTRO), Rua Simeão Camargo Varela de Sá, 03-Vila Carli, Guarapuava 85040-080,
`Paraná, Brazil
`* Correspondence: fsmurakami@ufpr.br; Tel.: +55-41-3360-4074
`
`2
`
`3
`
`Received: 14 June 2019; Accepted: 23 July 2019; Published: 24 July 2019
`
`Abstract: Sitagliptin is an inhibitor of the enzyme dipeptidyl peptidase-4, used for the treatment
`of type 2 diabetes mellitus. The crystal structure of active pharmaceutical solids determines their
`physical and chemical properties. The polymorphism, solvates and hydrates can influence the free
`energy, thermodynamic parameters, solubility, solid-state stability, processability and dissolution
`rate, besides directly affecting the bioavailability. Thus, the physicochemical characterization of
`an active pharmaceutical ingredient is required to guarantee the rational development of new
`dosage forms. In this context, we describe herein the solid-state characterization of three crystalline
`forms of sitagliptin: sitagliptin phosphate monohydrate, sitagliptin phosphate anhydrous and
`sitagliptin base form. The investigation was carried out using differential scanning calorimetry (DSC),
`thermogravimetry (TG)/derivative thermogravimetry (DTG), spectroscopic techniques, X-ray powder
`diffraction (XRPD) and morphological analysis by scanning electron microscopy. The thermal analysis
`◦
`revealed that during the dehydration of sitagliptin phosphate monohydrate (Tpeak = 134.43
`C,
`∆H = −1.15 J g
`−1) there is a characteristic crystalline transition event, which alters the physicochemical
`parameters of the drug, such as the melting point and solubility. The crystalline behavior of sitagliptin
`base form differs from that of sitagliptin phosphate monohydrate and sitagliptin phosphate anhydrous,
`mainly with regard to the lower temperature of the fusion event. The melting point (Tpeak) values
`◦
`◦
`obtained were 120.29
`C for sitagliptin base form, 206.37
`C for sitagliptin phosphate monohydrate
`◦
`and 214.92
`C for sitagliptin phosphate anhydrous. In relation to the thermal stability, sitagliptin
`phosphate monohydrate and sitagliptin phosphate anhydrous showed a slight difference; however,
`both are more thermostable than the base molecule. Therefore, through this study it was possible to
`establish the most suitable crystalline form of sitagliptin for the development of a safe, effective and
`appropriate pharmaceutical dosage form.
`
`Keywords: crystallinity; characterization in solid state; physicochemical properties; solubility;
`bioavailability
`
`1. Introduction
`
`Sitagliptin (Figure 1) is an inhibitor of the enzyme dipeptidyl peptidase-4 (DPP-4) and it
`consequently inhibits the degradation of incretin hormones, like glucagon-like peptide-1 (GLP-1) and
`
`Materials 2019, 12, 2351; doi:10.3390/ma12152351
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`
`materials
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`# & !*-
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`glucose-dependent insulinotropic peptide (GIP), resulting in increased insulin secretion and inhibited
`glucagon release by the beta and alpha cells of the pancreas, improving glycemic control [1,2].
`
`Figure 1. Chemical structure of sitagliptin phosphate monohydrate.
`
`Sitagliptin (7-[(3R)-3-amino-1-oxo-4-(2,4,5-trifluorophenyl)butyl]-5,6,7,8-tetrahydro-3-(trifluoromethyl)-
`1,2,4-triazolo[4,3-a]pyrazine) is an effective anti-glycemic drug used for the treatment of type 2 diabetes
`mellitus (T2DM) [3]. The original product is marketed by Merck & Co., under the trade name Januvia,
`as sitagliptin phosphate monohydrate, approved in 2006 by the Food and Drug Administration
`(FDA) [4,5].
`Sitagliptin phosphate monohydrate (STG) is white to off-white, crystalline, non-hygroscopic,
`soluble in water and N,N-dimethylformamide, slightly soluble in methanol and very slightly soluble
`in ethanol, acetone and acetonitrile. It has the molecular formula C16H15F6N5O·H3PO4·H2O and the
`−1 [6].molecular weight is 523.32 g mol
`
`The hydrate formation is of great importance in the pharmaceutical industry, considering the
`ubiquity of water vapor, because hydrates are frequently more stable [5]. Thus, the presence of solvates
`and hydrates influences the physicochemical properties of the crystals.
`A single molecule, such as sitagliptin, may give rise to a variety of crystalline forms with distinct
`crystal structures and physical properties. The structure of a crystal does not only determine the
`physicochemical properties of the active pharmaceutical ingredient (API) but also the solubility, stability,
`processability and bioavailability of the drug [7].
`Solubility is one of the most important parameters in relation to achieving the desired
`concentration of the drug in systemic circulation in order to obtain the required pharmacological
`response. Poorly water-soluble drugs with a slow absorption, for instance, may show inadequate
`bioavailability [8,9].
`The characterization of solid-state properties is a prerequisite in the development of new
`pharmaceutical solid dosage forms [5]. Thus, the characterization and differentiation of different
`crystalline forms of sitagliptin is required so that the physical properties of complex solvates and
`hydrates can be determined. Thermal analysis, for example, is a well-known technique for the
`characterization of APIs in terms of stability and structural investigations [10,11].
`Sitagliptin phosphate monohydrate is relatively characterized and commonly used in the
`pharmaceutical industry; however, little is known about other crystalline forms of sitagliptin, such as
`the anhydrous and base form, including the effect of the dehydration process and the production of
`base form on the physical and chemical stability of this pharmaceutical hydrate.
`Few patents such as US patent application 2015/0087834 A1 [12] reported a method for preparation
`of sitagliptin phosphate and sitagliptin phosphate anhydrous, providing a brief and superficial
`description of X-ray powder diffraction (XRPD) and differential scanning calorimetry (DSC). The US
`2009/0221595 A1 [13] reported the processes of preparing polymorphs forms, describing briefly X-ray
`powder diffraction and differential scanning calorimetry only the base form of sitagliptin. In fact, there
`is no studies in the literature that reports the detailed characterization these three crystalline forms.
`In this context, the novelty of this work aims to provide a detailed solid state characterization of
`STG, sitagliptin phosphate anhydrous (STGA) and sitagliptin base form (STGB). The physical-chemical
`characteristics were investigated using DSC, thermogravimetry (TG), non-isothermal kinetics analysis,
`
`
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`spectroscopic techniques (Fourier transform infrared (FTIR) and Raman), XRPD and scanning electron
`microscopy (SEM).
`
`2. Materials and Methods
`
`2.1. Sitagliptin Phosphate Monohydrate (STG)
`
`The active pharmaceutical ingredient, STG with 99.9% declared content, batch 20170904, was
`purchased from Baoji Guokang Bio-Technology (Baoji, China).
`
`2.2. Sitagliptin Phosphate Anhydrous (STGA)
`
`The sitagliptin anhydrous form was obtained by dehydration of sitagliptin phosphate monohydrate
`(STG). The process involved placing 2.5 g of STG in a vacuum oven (model 6030A VACUOTERM, São
`◦
`Paulo, Brazil) for 60 min at 300–400 mmHg and 150
`C.
`
`2.3. Sitagliptin Base Form (STGB)
`
`−1 of sitagliptin phosphateThe base form of sitagliptin was obtained by dissolving 0.008 mol g
`
`monohydrate (STG) in 92.10 mL of purified water and then adding ammonia (10%, v/v) at pH 10.0.
`The solution obtained was poured into a separatory funnel and washed twice with 83.73 mL of ethyl
`acetate. The organic phase was dried with anhydrous sodium sulfate and filtered through quantitative
`filter paper. The filtrate was evaporated in a rotary evaporator and then dried at room temperature in
`a vacuum desiccator. A white solid crystal powder was obtained with a yield of 92% [12].
`
`2.4. Differential Scanning Calorimetry (DSC)
`The DSC analysis was performed in a Shimadzu® DSC-60 calorimeter (Kyoto, Japan). Samples
`were analyzed in an aluminum crucible containing around 2.0 mg of sample under dynamic synthetic
`◦
`◦
`−1) with a heating rate of 10air atmosphere (50 mL min
`−1 and temperature range of 30C min
`
`
`C to
`◦
`400
`C. The equipment was calibrated with indium and zinc (reference standards).
`The purity was determined using aluminum crucibles with approximately 2.0 mg of sample at a
`◦
`◦
`◦
`−1 from 30C min
`heating rate of 2
`
`C to 400
`C. The purity of the sample was measured in triplicate
`using TASYS software (version 1.14, Shimadzu®), based on the Van’t Hoff equation:
`(To − Tm)∆H f
`RTo2
`
`X2 =
`
`(1)
`
`where the purity is determined from the molar percentage of impurities present in the sample, X2
`represents the mole fraction of impurities, Tm is the sample melting temperature, To is the melting point
`◦
`K), R is a gas constant and ∆Hf is the heat of fusion of the main component
`of the pure substance (
`−1) [14,15].(J mol
`
`
`2.5. Thermogravimetry (TG)
`The thermogravimetric analysis was carried out on a Shimadzu® DTG-60 thermal analyzer under
`−1. Approximately 4.0 mg of sample was placed in aa dynamic synthetic air atmosphere of 50 mL min
`
`◦
`◦
`◦
`−1.C min
`platinum crucible and heated from 30
`C to 400
`C at a heating rate of 10
`
`For the non-isothermal kinetics studies, the curves were obtained using five different heating
`−1. The kinetics parameters were determined by the Ozawa method using
`◦
`rates: 5, 10, 15, 20, 25
`C min
`TASYS software. The equipment was calibrated with calcium oxalate (reference standard).
`
`2.6. Thermogravimetry–Mass Spectrometry (TG-MS)
`
`Thermogravimetry coupled to mass spectrometry was used to study the thermal decomposition
`of sitagliptin phosphate monohydrate. The analysis was carried out on a TA Instruments®MS
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`Q600 SDT analyzer (New Castle, DE, USA). Approximately 2.0 mg of each sample was heated to
`◦
`◦
`−1) applying a heating rate of 10C under a dynamic nitrogen atmosphere (50 mL min
`−1.
`900
`
`C min
`The equipment generates separate TG curves and MS spectra, analyzing the mass/charge (m/z) with
`respect to temperature.
`
`2.7. Melting Point
`
`The melting behavior was determined using a Mettler-Toledo MP70 melting point system
`(Greifensee, Switzerland). A capillary was used with a closed bottom, applying a heating rate of
`◦
`◦
`−1 up to a temperature limit of 400C min
`10
`
`C.
`
`2.8. Fourier Transform Infrared (FTIR) Spectroscopy
`
`The infrared spectra were recorded on a Bruker Alpha-P FTIR spectrometer (Ettlingen, Germany)
`−1, with a nominalusing attenuated total reflection (ATR) in the wavelength range of 3500 to 500 cm
`
`−1 and accumulation of 32 scans.resolution of 4 cm
`
`
`2.9. Raman Spectroscopy
`
`The Raman spectra were obtained with a WITEC-Alpha 300R confocal Raman microscope (Ulm,
`−1, wavelength laser ofGermany), using a diode 3 mW source at a diffraction grating of 600 g mm
`
`−1 and accumulation of 30 scans.532 nm, integration time of 3 s, resolution of 5.0 cm
`
`
`2.10. X-ray Powder Diffraction (XRPD)
`The X-ray powder diffraction patterns were obtained on a Shimadzu® XRD-7000 X-ray
`diffractometer using a sample door of stainless steel of 20 mm with monochromatic radiation CuKα
`(λ = 1.5406 Å), a voltage of 40.0 kV, current of 20.0 mA, 2θ scanning angle and scan range of 2.0–40.0.
`
`2.11. Scanning Electron Microscopy (SEM)
`
`The morphological evaluation was performed by scanning electron microscopy (SEM) using a
`JEOL JSM-6360 LV microscope (São Paulo, Brazil). The sample was pre-metallized with gold and
`analyzed at low vacuum with an acceleration voltage of 15 kV at magnifications of 200×, 600×, 2000×
`and 5000×.
`3. Results and Discussion
`
`3.1. Thermal Characterization
`
`The thermal behavior of STG can be observed based on the DSC and TG/DTG curves in Figure 2.
`The DSC curve shows four endothermic events and six exothermic events. The first endothermic
`
`C; ∆H = −1.15 J g−1),
`◦
`◦
`C; Tonset = 126.76
`event corresponds to dehydration of the STG (Tpeak = 134.43
`
`C; ∆H = −42.00 J g−1),
`◦
`◦
`followed by a well-defined endothermic event (Tpeak = 142.30
`C; Tonset = 140.61
`corresponding to the crystalline transition of the sitagliptin phosphate, since no mass loss was
`◦
`◦
`C; Tonset = 203.02
`indicated by the TG/DTG curve. The third endothermic event (Tpeak = 206.37
`C;
`∆H = −104.97 J g
`−1) is characteristic of the melting process, and is in agreement with the melting point
`◦
`determined by capillary analysis (205.3
`C) and results reported in the literature [16]. Subsequent
`thermal events correspond to the decomposition process.
`◦
`◦
`C (∆m = 2.9%;
`The first mass loss on the TG/DTG curve at between 101
`C and 136
`◦
`DTGpeak = 117.20
`C) confirms the dehydration event observed on the DSC curve. The second
`mass loss event indicates that the thermal decomposition occurs in two stages immediately after the
`◦
`◦
`◦
`◦
`◦
`C (∆m = 7.9%; DTGpeak = 232.81
`fusion event, in the ranges of 192
`C to 243
`C) and 243
`C to 385
`C
`◦
`(∆m = 60.4%; DTGpeak = 300.57
`C). Thus, the TG/DTG curve confirms that there is thermal stability
`◦
`up to 192
`C STG.
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`The differential scanning calorimetry (DSC) and thermogravimetry/derivative
`Figure 2.
`thermogravimetry (TG/DTG) curves of sitagliptin phosphate monohydrate (STG) obtained with
`◦
`−1) and a heating rate of 10
`−1.
`a synthetic air atmosphere (50 mL min
`C min
`
`In order to compare the thermal behavior of the different crystalline forms of sitagliptin, the STGA
`was analyzed by DSC and TG and the results are shown in Figure 3.
`
`Figure 3. The DSC and TG/DTG curves for sitagliptin phosphate anhydrous (STGA) obtained with a
`◦
`−1) and a heating rate of 10
`−1.
`synthetic air atmosphere (50 mL min
`C min
`
`The DSC curve for STGA shows two endothermic events and three exothermic events. The first
`
`C; ∆H = −104.84 J g−1), which
`◦
`◦
`C; Tonset = 212.58
`is related to the melting point (Tpeak = 214.92
`◦
`was confirmed by the capillary method (212.2
`C). The subsequent events correspond to thermal
`decomposition, confirmed by the TG/DTG curve, indicating that thermal decomposition occurs in
`◦
`◦
`◦
`◦
`C (DTGpeak = 234.24
`C; DTGpeak = 278.74
`C; DTGpeak = 333.94
`three steps starting at 216
`C).
`
`
`
`
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`◦
`C seen on
`It can also be noted in Figure 3 that the first endothermic event between 125 and 145
`the STG curve is not present on the STGA curve and no mass loss is indicated by the TG/DTG analysis,
`confirming that the water of crystallization was no longer present in this molecule.
`Although the thermal profile of sitagliptin phosphate anhydrous (STGA) is similar to that
`of sitagliptin phosphate monohydrate (STG), there are some differences associated with the drug
`crystallinity, the STGA presenting a higher melting point and a decomposition profile with three stages.
`The DSC curve of the sitagliptin base form (STGB) in Figure 4 shows a single endothermic event,
`
`C; ∆H = −75.18 J g−1),
`◦
`◦
`C; Tonset = 117.81
`corresponding to the melting of the compound (Tpeak = 120.29
`◦
`C). The TG/DTG curve indicates that the thermal
`as confirmed by the capillary analysis (121.0
`◦
`◦
`C (∆m = 21.6%; DTGpeak = 244.53
`decomposition takes place in two steps, in the ranges of 193 to 272
`C),
`◦
`◦
`◦
`C (∆m = 61.8%; DTGpeak = 325.33
`and 272 to 367
`C), showing thermal stability up to 193
`C.
`
`Figure 4. The DSC and TG/DTG curves of sitagliptin base form obtained with a synthetic air atmosphere
`◦
`−1) and a heating rate of 10
`−1.
`(50 mL min
`C min
`
`On comparing STGB with the other forms of sitagliptin, it was noted that the thermal profiles
`◦
`differ, especially in relation to the fusion event. In fact, for the STGB form the melting point (120.29
`C)
`◦
`◦
`was significantly lower than the values for STGA (214.92
`C) and STG (206.37
`C). This result suggests
`different crystalline forms with changes in the crystal structure. A similar behavior was reported by
`Murakami et al. (2009) [10] who studied the different crystalline forms of omeprazole sodium.
`The analysis of the gases produced during thermal degradation can provide valuable information
`about the pathway of decomposition. Therefore, the thermogravimetry coupled with mass spectrometry
`(TG–MS) was used to analyze the gaseous products during the thermal degradation of STG.
`The TG–MS curve for STG, shown in Figure 5, provides information on the major volatile
`compounds released from sitagliptin phosphate monohydrate under controlled heating. The molecules
`most likely associated with the mass spectrum results are: H2O (m/z = 18), CO (m/z = 28) and C2H3N
`(m/z = 41).
`The thermal decomposition of organic molecules, theoretically, is due to the molecular kinetic
`energy increasing during heating process. These include atomic oscillations that rupture the weaker
`chemical bonds and the cleavage occurs easier with lower orders of chemical bonds. Thermodynamically,
`the decomposition process also depends on the stability of the decomposition products or intermediates
`generated [17]. In order to understand the thermal decomposition of STG, a theoretical discussion is
`made from the perspective of the molecular structure.
`
`
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`Figure 5. The thermogravimetric coupled with mass spectrometry (TG–MS) curves for sitagliptin
`−1), a heating rate of
`phosphate monohydrate obtained with synthetic air atmosphere (50 mL min
`◦
`−1 and 350 scans.
`10
`C min
`
`According to the molecular bond order distributions of STG, the position of the chemical bonds
`ruptured could be deduced, and the thermal decomposition mechanism in the thermal decomposition
`process could be proposed and is given in Figure 6.
`
`Figure 6. Proposal for the thermal decomposition of sitagliptin phosphate monohydrate.
`
`As seen from the results obtained in the thermogravimetric analysis, the initial mass loss for
`structure 1 is associated with dehydration of the compound (step A), which was confirmed by TG–MS,
`◦
`since m/z = 18 (H2O) has a higher intensity at 117
`C. Subsequently, in structure 2 can be seen the
`release of phosphate group (Step B).
`It can be seen from structure 3, in Figure 6, that the weakest bond of STG, no matter what kind of
`form, is the O=C–N bond, thus resulting in an intermediate structure 4 and 5 respectively. Therefore,
`the crucial step of thermal decomposition is the cleavage of the amide group as seen in step C. A similar
`fragmentation pattern was described by Vishnuvardhan et al. [18].
`◦
`Furthermore, at approximately 330
`C, it is possible to observe in step D, the probable cleavage of
`the C–C bond in structure 5, and the release of molecules such as CO (m/z = 28). Finally, in step E, the
`structure 6 has been associated with release of acetonitrile (CH3CN; m/z = 41), since it corresponds
`to the most stable form of stabilization when it assumes the cleavage between the C–ring and the
`
`
`
`F
`
`F
`
`F
`
`OH2
`
`+
`NH3
`O
`-
`H2PO4
`structure 1
`
`NN
`N
`
`F
`
`F
`
`F
`
`N
`
`step A
`
`F
`
`OH2
`(m/z = 18)
`
`F
`
`step E
`
`F
`
`F
`
`F
`
`NN
`N
`
`F
`
`F
`
`F
`
`N
`
`step B
`
`-
`
`H2PO4
`(m/z = 97)
`
`F
`
`F
`
`F
`
`+
`O
`NH3
`-
`H2PO4
`structure 2
`
`F
`
`F
`structure 7
`
`CH3CN
`(m/z = 41)
`
`F
`
`F
`structure 6
`
`step D
`
`NH2
`
`2 CO
`(m/z = 28)
`
`F
`
`F
`
`F
`
`O-
`
`+
`NH3
`
`O
`
`structure 5
`
`NN
`N
`
`F
`
`F
`
`F
`
`N
`
`O
`
`+
`NH3
`structure 3
`
`step C
`
`NN
`
`F
`
`N
`
`F
`
`F
`
`structure 4
`
`NH
`
`
`
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`branching of structure 6, resulting in a final structure 7. The thermal decomposition of the compound,
`occurring rapidly, successively and almost simultaneously, and this process appears as a three events
`on the TG/DTG curve.
`
`3.2. Purity Study
`
`The determination of purity is based on the assumption that an impurity modifies the characteristics
`of a pure material, changing the endothermic melting enthalpy. The melting transitions of a pure
`material (100% crystalline) must be sharp, and the presence of small amounts of impurities will
`generate a reduction in the melting point and widening of the overall melting range [19]. Figure 7
`shows the results for all samples and the calculated purity values were 99.16% ± 0.33 for sitagliptin
`phosphate monohydrate, 97.79 ± 0.87% for sitagliptin phosphate anhydrous and 98.32% ± 0.62 for
`sitagliptin base form.
`
`Figure 7. DSC curves for STG, STGA and sitagliptin base form (STGB) obtained with synthetic air
`◦
`−1.
`−1) and a heating rate of 2
`atmosphere (50 mL min
`C min
`
`Therefore, in this analysis, the melting events were observed as symmetrical and well-defined
`endotherms, indicating that the dehydration process and the production of the base form did not
`modify the purity characteristics.
`
`3.3. Kinetics Analysis
`
`In order to evaluate the stability of the STG, STGA and STGB forms, the samples were subjected to
`non-isothermal kinetics analysis based on five TG curves obtained at different heating rates. The effect
`of temperature was evaluated in terms of the thermal decomposition rate and reaction order.
`Non-isothermal thermogravimetric analysis with a linear heating rate is a technique for studying
`the process kinetics that allows the kinetics parameters of isoconversional solid-state reactions to
`be determined through calculations. The Flynn–Wall–Ozawa (FWO) method is one approach to
`studying these kinetics parameters, where the activation energy (Ea) is calculated by dynamic heat
`thermogravimetry [20,21].
`The isothermal method is based on the Arrhenius equation and is given by the following equation:
`log A = log[ZEa/Rf(α)] − 2.315 − 0.457(Ea/RT)
`
`(2)
`−1), R is
`where A is the heating rate, Z is the pre-exponential factor, Ea is the activation energy (J mol
`the gas constant; f(α) is the integral conversion function; and T is the temperature (K).
`
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`The natural logarithm of the heating rate (log A) against the inverse of the temperature (1/T) on
`the TG curves at different heating rates will give a straight line with a slope of −0.457 (Ea/R), allowing
`the determination of the activation energy [21–24].
`The kinetics results are shown in Figures 8–10. Overlapping the thermogravimetric curves
`revealed a shift to higher temperatures as the heating rate increases and the inserts in the figures show
`correlation curves with a linear trend for all samples.
`
`Figure 8. The TG curves for STG obtained for different heating rates with a synthetic air atmosphere (50
`−1). The inset shows the linear tendency of the correlation curves applying the Ozawa method.
`mL min
`
`Figure 9. The TG curves for STGA obtained for different heating rates with a synthetic air atmosphere (50
`−1). The inset shows the linear tendency of the correlation curves applying the Ozawa method.
`mL min
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`Figure 10. TG curves for STGB obtained for different heating rates with a synthetic air atmosphere
`−1). The inset shows the linear tendency of the correlation curves obtained applying the(50 mL min
`
`Ozawa method.
`
`The kinetics parameters obtained for STG (Figure 8), STGA (Figure 9) and STGB (Figure 10) are
`shown in Table 1.
`
`Table 1. Kinetics parameters obtained for STG, STGA and STGB in non-isothermal kinetics analysis.
`
`STG
`STGA
`STGB
`
`Activation Energy (Ea)
`89.29 ± 2.881 kJ mol
`−1
`88.84 ± 1.561 kJ mol
`−1
`78.21 ± 2.591 kJ mol
`−1
`
`Coefficient of Variation
`1.301%
`1.757%
`3.313%
`
`Reaction Order
`n = 0
`n = 0
`n = 0
`
`The results for the kinetics parameters demonstrate a slightly higher activation energy for STG,
`suggesting that the dehydration process had little effect on the thermal stability of the drug.
`Furthermore, the kinetics study shows that STGB has a significantly lower activation energy
`than STG and STGA, indicating that the removal of the phosphate group from sitagliptin reduces the
`thermal stability of the sample.
`
`3.4. Fourier Transform Infrared (FTIR) Spectroscopy
`
`The ATR-FTIR spectra obtained for the STG, STGA and STGB are shown in Figure 11. It can be
`observed that there are common vibrational bands for the three samples, which are associated with the
`major functional groups of sitagliptin.
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`Figure 11. Comparison of Fourier transform infrared (FTIR) spectra for samples of sitagliptin phosphate
`monohydrate (STG), sitagliptin phosphate anhydrous (STGA) and sitagliptin base form (STGB).
`−1 is related to alkane stretching (C–H),The band regions can be assigned as follows: 1465 cm
`
`
`−1 is associated with the C=O bond of carbonyl, and 1690–1640 cm−1 refers to the imine
`1750–1735 cm
`
`−1 is related to fluoride (C–F) and 3200–3100 cmgroup (C=N). The vibration at 1250–1000 cm −1 is
`
`associated with amine groups (NH2) [16,25–28].
`The main difference between STG and the other two samples (STGA and STGB) was seen in the
`−1, due to O–H stretching vibrations characteristic of water molecules,region between 3500 and 2500 cm
`
`present only in the sitagliptin phosphate monohydrate molecule [16,29].
`
`3.5. Raman Spectroscopy
`
`Raman spectroscopy is a technique based on the principle of dispersion where the emission of
`monochromatic laser radiation causes vibrations in the sample molecule from the inelastic collision of
`the incident monochromatic radiation (laser) and the molecules present in the sample [30].
`Raman spectroscopy was performed on the STG, STGA, STGB samples and the results are shown
`in Figure 12. Based on the spectra, the characterization of the different forms of sitagliptin can be
`
`−1, C–C at 1515–1525 cmbased on the stretching vibrations of C–H at 2950 cm −1, C=O at 1650 cm
`−1,
`
`−1 and N–H at 3077–3090 cm−1, along with the vibration related to the CF3 group at
`
`C–N at 1345 cm
`−1 [31].1025–1060 cm
`
`−1, present onlyThe major differences were observed in the O–H stretching vibrations at 3360 cm
`
`in the STG spectrum (related to water molecules) and the presence of the phosphate group (P–O)
`−1, present only in the case of the STG and STGA forms. In fact, thisstretching vibration at 815 cm
`
`result confirms the structure of the sitagliptin base form.
`
`
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`Figure 12. Raman spectra for sitagliptin phosphate monohydrate (STG), sitagliptin phosphate
`anhydrous (STGA) and sitagliptin base form (STGB).
`
`3.6. X-ray Powder Diffraction (XRPD)
`
`The diffraction of X-rays by a crystalline material following the direction of an incident X-ray
`beam toward a material with uniform atomic structure is caused by the constructive interference
`process. The XRPD technique was used to qualitatively identify the crystallinity of the samples [32].
`The XRPD results for STG, STGA and STGB are shown in Figure 13. According to the diffraction
`◦
`◦
`◦
`◦
`◦
`pattern, STG is a crystalline material, with major characteristic peaks at 13.2
`, 13.8
`, 15.9
`, 18.4
`, 19.1
`,
`◦ ± 0.2and 30.9
`◦
`◦
`◦
`◦
`◦
`◦
`21.2
`, 24.0
`, 25.0
`, 25.7
`, 29.5
`
`.
`◦
`◦
`,
`, 9.3
`The STGA sample also has crystalline character, the main characteristic peaks being at 4.6
`◦ ± 0.2and 26.9
`◦
`◦
`◦
`◦
`◦
`◦
`◦
`◦
`◦
`, 13.9
`, 15.0
`, 18.2
`, 19.2
`, 19.9
`, 21.4
`25.4
`
`. Dwivedi et al. (2015) observed similar
`13.4
`diffraction patterns for STG and STGA.
`The STGB showed several reflections corresponding to those described by Perlman (2009), as
`◦ ± 0.2and 28.8
`◦
`◦
`◦
`◦
`◦
`◦
`◦
`◦
`◦
`◦
`follows: 7.4
`, 11.5
`, 16.7
`, 17.7
`, 18.9
`, 24.1
`, 24.5
`, 27.0
`, 28.5
`
`.
`Comparatively, the STGA patterns showed reduced crystallinity after the dehydration process,
`leading to a slightly more unstable molecule, as observed in the kinetics analysis. However, the
`anhydrous molecule (STGA) had greater solubility when compared to STG.
`Based on the patterns obtained, the STGB has higher crystallinity, however, it has lower thermal
`stability, as observed in the thermal analysis, and lower solubility. Since a solvate is dissolved in water,
`free ions interact with the polar water molecules and these interactions provide pharmaceutical salts
`with greater solubility than the free form [33].
`
`
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`Figure 13. X-ray powder diffraction (XRPD) patterns for sitagliptin phosphate monohydrate (STG),
`sitagliptin phosphate anhydrous (STGA) and sitagliptin base form (STGB).
`
`The solubility is directly associated with the drug bioavailability, the aqueous solubility of a drug
`is a key property for the drug absorption after oral administration and thus low solubility leads to
`limited oral bioavailability [8,9].
`Considering that the synthetic route of STG generates solvates and hydrates [13], and the
`dehydration process is an expensive step in industrial scale. Thus, considering the slight difference
`between STG and STGA, the most suitable form for pharmaceutical purposes and the development of
`dosage forms is STG.
`
`3.7. Scanning Electron Microscopy (SEM)
`
`Analysis by scanning electron microscopy (SEM) provides information on the sample morphology,
`including crystal size and shape, which has a direct influence on the physical characteristics and may
`influence in pharmaceutical operations and mechanical properties, such as processability (grinding,
`mixing, powder flow, compression, dissolution and lyophilization) [34].
`The micrographs for STG, STGA and STGB obtained by SEM are shown in Figure 14.
`The sitagliptin phosphate monohydrate shown in Figure 14(A1,A2) can be classified as an
`orthorhombic crystal system. As described by Kaduk et al.
`(2015) [35], the structure of STG is
`comprised of flat and thin crystals (platy form).
`The microscopic evaluation of sitagliptin phosphate anhydrous (Figure 14(B1,B2)) revealed that
`the dehydration process did not alter the morphological characteristics compared with STG. The images
`C1 and C2 in Figure 14 show that the particles of the sitagliptin base form were smaller (by around a
`factor of 10) when compared to the other samples (note the magnification), but maintained the same
`shape and morphology.
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`Figure 14. Photomicrographs obtained by scanning electron microscopy (SEM): STG (A1 and A2; 200×
`and 600×), STGA (B1 and B2; 200× and 600×) and STGB (C1 and C2; 2000× and 5000×).
`4. Conclusions
`
`The solid-state characterization of sitagliptin phosphate monohydrate (STG), sitagliptin phosphate
`anhydrous (STGA) and the sitagliptin base form (STGB) was carried out using several analytical
`techniques, such as DSC, TG, FTIR and RAMAN spectroscopy, XRPD and SEM.
`The thermoanalysis showed that after the dehydration of STG there was a crystalline transition,
`altering the melting point and solubility of the compound. The dehydration of STG was confirmed by
`◦
`TG–MS analysis, with the release of water at 117
`C.
`The crystalline behavior of the sitagliptin base form (STGB) differed from that of the STG and
`◦
`STGA, and the major difference was a lower fusion event. Specifically, the melting points were 120.29
`C
`◦
`◦
`C for STG and 214.92
`C for STGA.
`for STGB, 206.37
`During pre-formulation studies in the development of new dosage forms, it is essential