# FT-IR Spectroscopy Applied for Identification of a Mineral Drug Substance in Drug Products: Application to Bentonite

Authors: H. Ouhaddouch; A. Cheikh; M. O. B. Idrissi; M. Draoui; M. Bouatia
Journal: Journal of Spectroscopy
 (2019)

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## Abstract

The aim of this study is to prove the effectiveness of IR spectroscopy as an identification test able to discriminate between mineral compounds in mixtures. This work is concerned with the physical characterisation of purified bentonite, bentonite in organic mixtures and organic excipients, and mineralized organic mixture containing bentonite using FT-IR spectroscopy. The different spectra were compared with each other in order to determine fingerprints of bentonite represented by bands located at 3632 cm−1 and 3437 cm−1. The analysis of the spectra of the nonmineralized mixture demonstrates the presence of two bands at 1454 and 2928 cm−1, superimposed on those of the excipients and which disappear after 2 hours of mineralization at 500°C. Finally, we notice a displacement of the stretching band of H2O to the right with increasing the proportion of the excipients.


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## Body

## 1. Introduction

Over the last decade, infrared (IR) spectroscopy had proved to be a powerful analytical method widely applied in quality control in the field of agricultural, environment, food, and especially pharmaceutical. It is the best technique used in the investigation and identification of clays and clay minerals, especially bentonite, by a combination of spectroscopic and spatial information [1, 2]. Using this method, selected sample areas can be analyzed with reference to the identification and localization of chemical species by Fourier-transform infrared spectroscopy (FT-IR) in the transmission or attenuated total reflection (ATR) mode [3].On the other hand, FT-IR is an excellent technique for pharmaceutical analysis which offers many advantages since it is easy to use, sensitive, selective, green, and fast (the total analysis time including making the pellets, measurement, identification, and report generation is lower than 10 minutes) and helps ensure regulatory compliance through validation protocols. Contrary to high-performance liquid chromatography (HPLC) which is less fast, requires the preliminary preparation of the mobile phase, and is not applied in the field of mineral chemistry, this spectroscopic method is the reference for identification of organic drug substances in pharmacopeia. The IR spectroscopy is mainly complementary to X-ray diffraction (XRD) and other methods used to study clays.The interpretation of the different IR spectra remains empirical and consists most often in comparing the results obtained with previously recorded reference spectra or to put in evidence important structural parts of the molecules with intense vibration bands [4–6], even if the substance is in mixtures or complexes [7]. It is based mainly on the analysis of IR spectra of isolated molecules. However, since these spectra can serve as fingerprints for identification, we are interested in proving the effectiveness of this spectral method in the identification of a mineral product that cannot be detected by HPLC in a mixture.The aim of this study is to develop a new method for identification of the drug substance as purified bentonite in a drug product, using FT-IR spectroscopy in order to apply it in several fields such as industrial pharmacy to analyze mineral intestinal adsorbents [8].


## 2. Materials and Methods

### 2.1. Preparation of Mixture

The starting material is bentonite clay, which belongs to the family of crystalloids. Five mixtures of drug products were prepared of each proportion of bentonite (Sigma-Aldrich, analytical grade), pure glucose monohydrate (Riedel-de Haën, analytical grade), and menthol (BASF, pharmaceutical grade) (Table1). The mixtures thus formed are triturated in a porcelain mortar to reduce the average particle size to 1-2 μm in order to promote the homogeneity of the samples [9]. In effect, the size of the particles influences the amount of absorption of the sample and the intensity of the absorption peaks of its spectrum. The difference in particle size between the different constituents of the mixture then complicates the analysis of the mixture spectra in which the coarsest compound becomes predominant [10].Table 1
Composition of the studied mixtures.
Code
Mixtures
Bentonite (mg)
Glucose (mg)
Menthol (mg)
M80
Mixture 80%
80
19
1
M60
Mixture 60%
60
39
1
M40
Mixture 40%
40
59
1
B
Bentonite
100
—
—
G
Glucose
—
100
—The aim is to have mixtures sufficiently simple to acquire a knowledge of the interaction spectra related to the superposition of the absorption spectra of the different species, knowing that the additivity of the absorbances is an ideal case. The mixtures thus prepared will be read after by IR spectroscopy.


### 2.2. Mineralization of the Mixture

The determination of the time required for the complete mineralization of glucose and menthol (excipients) led us to prepare six samples of mixture 80% (M80) and one sample of purified bentonite (B). Each 100 mg sample was placed in a porcelain crucible at a temperature of 500°C in a temperature-controlled oven of the Conacom Italia type. The crucibles of the mixture were recovered one after the other every 30 minutes for 3 hours, in order to be analyzed by FT-IR spectroscopy using the transmission mode.


### 2.3. Measurement by Fourier-Transform Infrared Spectroscopy (FT-IR)

The FT-IR spectroscopy using the transmission mode is an intuitive method which does not require sophisticated sampling accessories. The sample can be placed directly into the path of the infrared beam (with the help of a sample holder), and 128 scans were collected with a resolution of 4 cm−1 for each measurement over the spectral range of 500–4000 cm−1.FT-IR spectroscopy using KBr-pressed disk technique was conducted on a JASCO FT-IR 460 PLUS spectrometer (Pike Technologies, Madison, USA) equipped with a pyroelectric DLATGS detector. 2.5 mg of each mineralized sample and 100 mg of potassium bromide (Honeywell Fluka, infrared grade) were weighted, ground in an agate mortar, and pressed for 2 minutes at 10 tones/cm2 to form a semitransparent pellet which lets light to be transmitted to the detector [11, 12]. The pellet was placed in the IR beam using the sample transmission holder. The samples analyzed were nonmineralized glucose (G), purified bentonite (B), mineralized bentonite (MB), nonmineralized mixture 80% (M80), mineralized mixture 80% (MM80), nonmineralized mixture 60% (M60), and nonmineralized mixture 40% (M40). Three measurements were carried out in the transmission mode for each sample. Spectra Manager II spectroscopy software developed by JASCO ensures spectral acquisition and processing.The first part of this work was reserved to record the spectra of mineralized mixture MM80 for different durations in the aim to determine the time required for the complete mineralization of glucose and menthol (excipients). The second part of this work is to identify functional groups of nonmineralized mixture by measuring the absorption at specific wavelengths of bonds that vibrate independently of one another in order to confirm that the FT-IR spectroscopy is the most efficient method able to discriminate between different compounds of drug product.


## 2.1. Preparation of Mixture

The starting material is bentonite clay, which belongs to the family of crystalloids. Five mixtures of drug products were prepared of each proportion of bentonite (Sigma-Aldrich, analytical grade), pure glucose monohydrate (Riedel-de Haën, analytical grade), and menthol (BASF, pharmaceutical grade) (Table1). The mixtures thus formed are triturated in a porcelain mortar to reduce the average particle size to 1-2 μm in order to promote the homogeneity of the samples [9]. In effect, the size of the particles influences the amount of absorption of the sample and the intensity of the absorption peaks of its spectrum. The difference in particle size between the different constituents of the mixture then complicates the analysis of the mixture spectra in which the coarsest compound becomes predominant [10].Table 1
Composition of the studied mixtures.
Code
Mixtures
Bentonite (mg)
Glucose (mg)
Menthol (mg)
M80
Mixture 80%
80
19
1
M60
Mixture 60%
60
39
1
M40
Mixture 40%
40
59
1
B
Bentonite
100
—
—
G
Glucose
—
100
—The aim is to have mixtures sufficiently simple to acquire a knowledge of the interaction spectra related to the superposition of the absorption spectra of the different species, knowing that the additivity of the absorbances is an ideal case. The mixtures thus prepared will be read after by IR spectroscopy.


## 2.2. Mineralization of the Mixture

The determination of the time required for the complete mineralization of glucose and menthol (excipients) led us to prepare six samples of mixture 80% (M80) and one sample of purified bentonite (B). Each 100 mg sample was placed in a porcelain crucible at a temperature of 500°C in a temperature-controlled oven of the Conacom Italia type. The crucibles of the mixture were recovered one after the other every 30 minutes for 3 hours, in order to be analyzed by FT-IR spectroscopy using the transmission mode.


## 2.3. Measurement by Fourier-Transform Infrared Spectroscopy (FT-IR)

The FT-IR spectroscopy using the transmission mode is an intuitive method which does not require sophisticated sampling accessories. The sample can be placed directly into the path of the infrared beam (with the help of a sample holder), and 128 scans were collected with a resolution of 4 cm−1 for each measurement over the spectral range of 500–4000 cm−1.FT-IR spectroscopy using KBr-pressed disk technique was conducted on a JASCO FT-IR 460 PLUS spectrometer (Pike Technologies, Madison, USA) equipped with a pyroelectric DLATGS detector. 2.5 mg of each mineralized sample and 100 mg of potassium bromide (Honeywell Fluka, infrared grade) were weighted, ground in an agate mortar, and pressed for 2 minutes at 10 tones/cm2 to form a semitransparent pellet which lets light to be transmitted to the detector [11, 12]. The pellet was placed in the IR beam using the sample transmission holder. The samples analyzed were nonmineralized glucose (G), purified bentonite (B), mineralized bentonite (MB), nonmineralized mixture 80% (M80), mineralized mixture 80% (MM80), nonmineralized mixture 60% (M60), and nonmineralized mixture 40% (M40). Three measurements were carried out in the transmission mode for each sample. Spectra Manager II spectroscopy software developed by JASCO ensures spectral acquisition and processing.The first part of this work was reserved to record the spectra of mineralized mixture MM80 for different durations in the aim to determine the time required for the complete mineralization of glucose and menthol (excipients). The second part of this work is to identify functional groups of nonmineralized mixture by measuring the absorption at specific wavelengths of bonds that vibrate independently of one another in order to confirm that the FT-IR spectroscopy is the most efficient method able to discriminate between different compounds of drug product.


## 3. Results and Discussion

### 3.1. Mineralization of the Mixture

Disappearance of functional groups during the process of mineralization can be successfully monitored by transmission FT-IR spectrometry. The focus was on the band located at 2928 cm−1 corresponding to C-H stretching which is specific to organic compounds: glucose and menthol. The IR spectra of the mixture M80 mineralized at 500°C for 30, 60, 90, and 120 min (Figures 1 and 2) show a gradual decrease in the intensity of this band, until disappearance from the second hour (crucibles 4, 5, and 6). The disappearance of this peak testifies to a complete mineralization of the organic matter used in the preparation of the mixture (glucose and menthol). Consequently, the MM80 mixture used in our study represents a M80 mixture mineralized at 500°C for 2 hours.Figure 1
FT-IR spectra of nonmineralized mixture 80% (M80) mineralized at 500°C for different durations.Figure 2
Spectral band characteristic of nonmineralized mixture 80% (M80) after mineralization at 500°C for 30, 60, 90, and 120 minutes.


### 3.2. Data Processing of the Infrared Spectrometry

IR spectroscopy analysis of the different mixtures studied requires an understanding of the absorption bands attributable to the different physical and chemical properties of the material and which are used to assist in the identification of the various compounds that make up the mixture.It is well known that molecule analyzed by IR spectroscopy absorbs only the frequencies of IR light that match vibrations that cause a change in the dipole moment of the molecule. Every molecule, with the exception of enantiomers, has a unique infrared spectrum. This is due to the fact symmetrical structures and identical groups at each end of one bond will not absorb in the IR range [13]. The spectrum has two regions. The fingerprint region is unique for a molecule, and the functional group region is identical for molecules with the same functional groups [13].The FT-IR spectral examination of purified bentonite revealed different bands (Table2) comparable to those defined by the literature [14–18]. Two bands of different intensities, located at 3632 cm−1 and 3437 cm−1, also defined by Jeddi et al. [19], constitute the spectral signature specific to bentonite which allows its identification in a mixture of species. These bands correspond, respectively, to the vibrational modes of hydroxyl groups and water molecules absorbed in the interstitial spaces of the bentonite.Table 2
IR bands characteristic of the purified bentonite spectrum.
Transmission vibrational frequency (cm−1)
Strength
Functional groups
3632
Medium
OH stretching
3437
Medium
H2O stretching
1638
Weak
H2O bending
1042
Broad
Si-O stretching
915
Broad
Al-OH-Al bending
884
Medium
Al-OH-Fe bending
842
Medium
Al-OH-Mg bending
795
Weak
Si-O stretching of quartz and silica
622
Weak
Al-O + Si-O out-of-plane vibration
522
Broad
Al-O-Si bendingAccording to the spectra of the mineralized bentonite and purified bentonite obtained (Figure3), the general appearance is similar, but we can see a significant decrease in the intensity of the band at 3437 and 1638 cm−1 related to H2O absorbed on the samples, and a well-resolved band at 3632 cm−1 assigned to OH− stretching vibrations of structural hydroxyls remained [20]. This change of intensity means that the hydrophilicity of the bentonite decreased.Figure 3
FT-IR spectra of purified bentonite (B) and mineralized bentonite (MB).Figure4 shows FT-IR spectra of the mineralized bentonite and the excipients as well as those of the mixtures M80 and MM80. The different spectra were compared with each other in order to obtain information on the differences between the mineralized bentonite and the mixtures M80 and MM80. The spectra of the mixture MM80 are completely confused with those of the mineralized bentonite. However, those of the mixture M80, unlike the mixture MM80, have in the field ranging from 1300 to 4000 cm−1 and in addition to the bands characteristic of the hydroxyl group (3632 cm−1) and water (3437, 1638 cm−1), a small band at 1454 cm−1 and a sharper band at 2928 cm−1. This supports the additivity of absorbances of the excipients and the bentonite.Figure 4
FT-IR spectra of mineralized bentonite (MB) and mineralized mixture 80% (MM80) versus those of nonmineralized mixture 80% (M80) and excipients.The FT-IR spectroscopy of the mixtures M80, M60, M40, and excipients shown in Figure5 was carried out for a comparative purpose in order to obtain information about the modification of the mixture spectra while increasing the proportion of the excipients. The position, the shape, and the intensity of the stretching band of H2O at 3437 cm−1 were influenced by the addition of the excipients. Indeed, we notice a displacement of this band to the right with increase in its intensity while going from the mixture M80 to the mixture M40. The absorption bands observed at 1454 and 2928 cm−1 in the FT-IR spectra of the M80 mixture increase their intensity with increasing concentration of the excipients.Figure 5
FT-IR spectra of mineralized mixture 80% (MM80), nonmineralized mixture 80% (M80), nonmineralized mixture 60% (M60), nonmineralized mixture 40% (M40), and excipients.Unlike to FT-IR spectroscopy which is a simple, fast, cheaper, and extremely useful for the characterisation of both organic products and inorganic products, the HPLC is a method which represents some limitations rarely discussed that promotes the utilisation of FT-IR spectroscopy.The HPLC requires a sophisticated instrumentation with bewildering number of modules, columns, and mobile phases, operating parameters render HPLC difficult for the novice, and it is relatively expensive [21]. It can also be time-consuming, tedious, arduous, involve extensive chemical use, need regular maintenance requirements, and sometime, require pretreatment of samples [21, 22]. But, the main drawbacks of HPLC are the lack of a high-sensitivity universal detector and the insufficient chromatographic efficiency to separate many complexes like the inorganic products [22–24].Therefore, we can confirm that the FT-IR spectroscopy, applied to study drug product, is a very sensitive technique that provides a relatively easy and fast way to determine an unknown mineral drug substance, by giving detailed qualitative information on chemical composition of the analyzed material along with a unique fingerprint that often enables confirmation of its identity [25]. This identification and recognition of its presence in mixtures are more certain when its absorption bands are numerous and sharply defined and if there appears a distinctive region of the spectrum.


## 3.1. Mineralization of the Mixture

Disappearance of functional groups during the process of mineralization can be successfully monitored by transmission FT-IR spectrometry. The focus was on the band located at 2928 cm−1 corresponding to C-H stretching which is specific to organic compounds: glucose and menthol. The IR spectra of the mixture M80 mineralized at 500°C for 30, 60, 90, and 120 min (Figures 1 and 2) show a gradual decrease in the intensity of this band, until disappearance from the second hour (crucibles 4, 5, and 6). The disappearance of this peak testifies to a complete mineralization of the organic matter used in the preparation of the mixture (glucose and menthol). Consequently, the MM80 mixture used in our study represents a M80 mixture mineralized at 500°C for 2 hours.Figure 1
FT-IR spectra of nonmineralized mixture 80% (M80) mineralized at 500°C for different durations.Figure 2
Spectral band characteristic of nonmineralized mixture 80% (M80) after mineralization at 500°C for 30, 60, 90, and 120 minutes.


## 3.2. Data Processing of the Infrared Spectrometry

IR spectroscopy analysis of the different mixtures studied requires an understanding of the absorption bands attributable to the different physical and chemical properties of the material and which are used to assist in the identification of the various compounds that make up the mixture.It is well known that molecule analyzed by IR spectroscopy absorbs only the frequencies of IR light that match vibrations that cause a change in the dipole moment of the molecule. Every molecule, with the exception of enantiomers, has a unique infrared spectrum. This is due to the fact symmetrical structures and identical groups at each end of one bond will not absorb in the IR range [13]. The spectrum has two regions. The fingerprint region is unique for a molecule, and the functional group region is identical for molecules with the same functional groups [13].The FT-IR spectral examination of purified bentonite revealed different bands (Table2) comparable to those defined by the literature [14–18]. Two bands of different intensities, located at 3632 cm−1 and 3437 cm−1, also defined by Jeddi et al. [19], constitute the spectral signature specific to bentonite which allows its identification in a mixture of species. These bands correspond, respectively, to the vibrational modes of hydroxyl groups and water molecules absorbed in the interstitial spaces of the bentonite.Table 2
IR bands characteristic of the purified bentonite spectrum.
Transmission vibrational frequency (cm−1)
Strength
Functional groups
3632
Medium
OH stretching
3437
Medium
H2O stretching
1638
Weak
H2O bending
1042
Broad
Si-O stretching
915
Broad
Al-OH-Al bending
884
Medium
Al-OH-Fe bending
842
Medium
Al-OH-Mg bending
795
Weak
Si-O stretching of quartz and silica
622
Weak
Al-O + Si-O out-of-plane vibration
522
Broad
Al-O-Si bendingAccording to the spectra of the mineralized bentonite and purified bentonite obtained (Figure3), the general appearance is similar, but we can see a significant decrease in the intensity of the band at 3437 and 1638 cm−1 related to H2O absorbed on the samples, and a well-resolved band at 3632 cm−1 assigned to OH− stretching vibrations of structural hydroxyls remained [20]. This change of intensity means that the hydrophilicity of the bentonite decreased.Figure 3
FT-IR spectra of purified bentonite (B) and mineralized bentonite (MB).Figure4 shows FT-IR spectra of the mineralized bentonite and the excipients as well as those of the mixtures M80 and MM80. The different spectra were compared with each other in order to obtain information on the differences between the mineralized bentonite and the mixtures M80 and MM80. The spectra of the mixture MM80 are completely confused with those of the mineralized bentonite. However, those of the mixture M80, unlike the mixture MM80, have in the field ranging from 1300 to 4000 cm−1 and in addition to the bands characteristic of the hydroxyl group (3632 cm−1) and water (3437, 1638 cm−1), a small band at 1454 cm−1 and a sharper band at 2928 cm−1. This supports the additivity of absorbances of the excipients and the bentonite.Figure 4
FT-IR spectra of mineralized bentonite (MB) and mineralized mixture 80% (MM80) versus those of nonmineralized mixture 80% (M80) and excipients.The FT-IR spectroscopy of the mixtures M80, M60, M40, and excipients shown in Figure5 was carried out for a comparative purpose in order to obtain information about the modification of the mixture spectra while increasing the proportion of the excipients. The position, the shape, and the intensity of the stretching band of H2O at 3437 cm−1 were influenced by the addition of the excipients. Indeed, we notice a displacement of this band to the right with increase in its intensity while going from the mixture M80 to the mixture M40. The absorption bands observed at 1454 and 2928 cm−1 in the FT-IR spectra of the M80 mixture increase their intensity with increasing concentration of the excipients.Figure 5
FT-IR spectra of mineralized mixture 80% (MM80), nonmineralized mixture 80% (M80), nonmineralized mixture 60% (M60), nonmineralized mixture 40% (M40), and excipients.Unlike to FT-IR spectroscopy which is a simple, fast, cheaper, and extremely useful for the characterisation of both organic products and inorganic products, the HPLC is a method which represents some limitations rarely discussed that promotes the utilisation of FT-IR spectroscopy.The HPLC requires a sophisticated instrumentation with bewildering number of modules, columns, and mobile phases, operating parameters render HPLC difficult for the novice, and it is relatively expensive [21]. It can also be time-consuming, tedious, arduous, involve extensive chemical use, need regular maintenance requirements, and sometime, require pretreatment of samples [21, 22]. But, the main drawbacks of HPLC are the lack of a high-sensitivity universal detector and the insufficient chromatographic efficiency to separate many complexes like the inorganic products [22–24].Therefore, we can confirm that the FT-IR spectroscopy, applied to study drug product, is a very sensitive technique that provides a relatively easy and fast way to determine an unknown mineral drug substance, by giving detailed qualitative information on chemical composition of the analyzed material along with a unique fingerprint that often enables confirmation of its identity [25]. This identification and recognition of its presence in mixtures are more certain when its absorption bands are numerous and sharply defined and if there appears a distinctive region of the spectrum.


## 4. Conclusions

The present study clearly demonstrates that FT-IR spectroscopy is an efficient method for the characterisation of the purified bentonite in an organic mixture. This technique remains economical, rapid, and specific. The spectrum can be obtained in a few minutes using the inexpensive instruments, which can be available in many analytical laboratories and can be served as a fingerprint for new drugs of mineral origin as an alternative to chromatographic retention time in HPLC.Indeed, the FT-IR spectroscopy can be combined with other spectral methods such as X-ray diffraction in order to increase the specificity of the technique.



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*Source: 2960845-2019-03-03.xml*