http://informahealthcare.com/ddi ISSN: 0363-9045 (print), 1520-5762 (electronic) Drug Dev Ind Pharm, 2015; 41(1): 63–69 ! 2015 Informa Healthcare USA, Inc. DOI: 10.3109/03639045.2013.845842
RESEARCH ARTICLE
A study of photostability and compatibility of the anti-chagas drug Benznidazole with pharmaceutics excipients
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Fabiana L. A. Santos1, Larissa A. Rolim1, Camila B. M. Figueireˆdo1, Magaly A. M. Lyra1, Monize S. Peixoto1, Leslie R. M. Ferraz1, Jose´ L. Soares-Sobrinho1, A´dley Antonini Neves de Lima1,2, Ana Cristina Lima Leite3, and Pedro Jose´ Rolim Neto1 1
Laborato´rio de Tecnologia dos Medicamentos, Departamento de Cieˆncias Farmaceˆuticas, Universidade Federal de Pernambuco, Rua Prof. Arthur de Sa´, s/n, Cidade Universita´ria, Recife, PE, Brazil, 2Laborato´rio de Tecnologia Farmaceˆutica, Faculdade de Cieˆncias Farmaceˆuticas, Universidade Federal do Amazonas, Rua Alexandre Amorim, no. 330, Aparecida, Manaus, AM, Brazil, and 3Laborato´rio de Planejamento, Avaliac¸a˜o e Sı´ntese de Fa´rmacos, Departamento de Cieˆncias Farmaceˆuticas, Universidade Federal de Pernambuco, Rua Prof. Arthur de Sa´, s/n, Cidade Universita´ria, Recife, PE, Brazil Abstract
Keywords
Context: Benznidazole (BNZ) is an antiparasitic with trypanocidal properties for the etiological treatment of Chagas disease since 1973. Monitoring the stability of this drug is one of the most effective methods of assessment, forecasting and prevention of problems related to quality product. Objective: To investigate the direct and indirect photodegradation of BNZ and to evaluate the interference of the excipients used in the forms dosage solid as well as to shed light on the chemical structure of the degradation products obtained. Materials and methods: To perform this work we adopted the ‘‘ICH Harmonised Tripartite Guideline: Photostability Testing of New Drug Substances and Products Q1B’’ (Guideline Q1B). We used benzonidazole (BNZ) (N-benzil-2-(2-nitroimidazol-1-il) acetamide) (LAFEPEÕ , Recife, Brazil) and various excipients; beyond high-performance liquid chromatography (HPLC), differential scanning calorimetry (DSC), infrared spectroscopy (IR) and mass spectrometry/mass spectrometry (MS/MS). The indirect photodegradation of BNZ was carried out using physical mixtures with 13 pharmaceutical excipients commonly used in the preparation of solid dosage forms. Results: HPLC and MS/MS techniques were selected for the identification of two photoproducts (PPs) and photoreactions found in direct and indirect tests with the microcrystalline cellulose, considered a critical excipient. Discussion: Despite variations in the infrared spectrometry, differential scanning calorimetry and differential thermogravimetry curves, these techniques are not conclusive since the study of photodegradation of the drug caused decay of 30%, according to the ICH. Conclusions: The results show that BNZ only undergoes direct photodegradation, since no new PPs were found for a combination of the drug and excipients.
Benznidazole, Chagas disease, degradation product, HPLC-MS/MS, photodegradation
Introduction Endemic in 21 Latin American countries, Chagas disease kills every year, more people in the region than any other parasitic disease, including malaria1. Benznidazole (N-benzyl-2-nitro-1imidazolacetamide) is an antiparasitic, nitroimidazole derivative with trypanocidal properties, which has been used since 1973 for the etiological treatment of Chagas disease2. Two of the principal problems with this drug are its toxicity and low solubility3.
Address for correspondence: Fabiana Lı´cia Arau´jo dos Santos, Laborato´rio de Tecnologia dos Medicamentos, Departamento de Cieˆncias Farmaceˆuticas, Universidade de Pernambuco. Rua Professor Arthur de Sa´, s/n, Cidade Universita´ria, 50740-521 Recife, PE, Brasil. Tel: +55(81) 3272-1383. E-mail: [email protected]
History Received 11 July 2013 Revised 6 September 2013 Accepted 9 September 2013 Published online 17 October 2013
Despite the importance of treatment of the disease Benznidazole, few studies discuss the stability of the drug in the presence of light. In 2010, Soares-Sobrinho4 conducted a compatibility study during a tablet preformulation study and compared with the commercially available tablet, but he did not assess the formation of photodegradation products. Monitoring the stability of drugs is one of the most effective methods of assessment, forecasting and prevention of problems related to quality product during its validity. The safety and efficacy can also be evaluated by monitoring the formation of degradation products that may cause loss of therapeutic activity or toxicity5. In a situation where the isolated drugs absorb ultraviolet or visible radiation and this results in the loss of the drug and the formation of degradation products, there is a direct photoreaction. In the case of indirect photoreactions, it is not the drug, but another component of the formulation (e.g. excipients or impurities) that
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absorb ultraviolet or visible radiation, resulting in the formation of one or more reactive species which react with the drugs6–8. Thus, the aim of this study was to investigate the direct and indirect photodegradation of BNZ in solid state and to shed light on the chemical structure of the degradation products obtained, to evaluate the interference of the excipients used in the formulation commercially available and other excipients which may be used to obtain pharmaceutical forms that can solve the problems of solubility of the drug. To perform this work we adopted the ‘‘ICH Harmonised Tripartite Guideline: Photostability Testing of New Drug Susbtances and Products Q1B’’ (Guideline Q1B)9.
Material and methods
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Materials BNZ 99.9% (purity determined by high performance liquid chromatography (HPLC), using methods developed by Silva in 2007)10, N-benzil-2-(2-nitroimidazol-1-il)acetamide, raw material, and the standard were provided by Laborato´rio Farmaceˆutico do Estado de Pernambuco (LAFEPEÕ , Recife, Brazil), donated by RocheÕ Pharmaceutical Industry. All solvents and other substances used were of analytical grade. The solutions were prepared using ultrapure water (Milli QÕ , Billerica, MA) and filtered through 0.22 mm porosity filter membranes (MilliporeÕ , Billerica, MA). The excipients used were from various commercial sources: cellulose microcristaline, croscarmellose sodium, sodium starch glycolate and magnesium stearate (Blanver and MicrocelÕ Ltd, Itapevi, Brazil), starch 1500Õ and hydroxypropylmetilcellulose (HPMC) (ColorconÕ , West Point, MS), lactose (MeggleÕ , Wasserburg, Germany), talc (MagnesitaÕ , Contagem, Brazil), hydroxypropylcellulose (HPC) (Aqualon, Wilmington, DE), ethylcellulose, polyethylenoglycol (PEG 4000), polyvinylpirrolidone – PVP K30 (ISP CorpÕ , Miami, FL) and colloidal silicon dioxide (HenkelÕ , Du¨sseldorf, Brazil). Analytical method indicative of stability The BNZ assay was performed using HPLC on a ShimadzuÕ (Kyoto, Japan) with a controller system (Shimadzu SCL – 20VP), pumps A and B (Shimadzu LC – 20AD VP), a PDA detector (SPDM20A VP), auto-injector (Shimadzu SIL – 20AD VP), and oven (Shimadzu CTO – 20A) and the chromatograms were drawn up using the LC program – SolutionÕ 1.0. The following analytical conditions were used: column reverse phase ShimadzuÕ , C18, 5 mm, 25 cm 0.46 cm, flow 1.0 mL min1, injection volume: 40 mL, a wavelength of 210 nm, temperature 40 C and mobile phase: acetonitrile – phosphate buffer (pH 2.7; 20 mM) (80:20, v/v). Validation analytical method In the process of validation of analytical methods developed were assessed the following parameters: robustness, linearity, range, detection limit, quantification limit, precision, accuracy and selectivity11. Each sample, followed by a standard curve, was prepared from a stock solution daily, in concentrations of 8, 12 and 16 mg/mL containing the BNZ and their chemical reference substances (CRS): sulfate aminoimidazol, 2-nitroimidazole (2-NIZ) and N-benzilcloroacetamida (NBCA) simultaneously. In the robustness test variables were analyzed: the manufacturer of the dibasic potassium phosphate (mobile phase component), evaluating the following marks: Carlo ErbaÕ (Rodano, Italy) and SinthÕ (Diadema, Brazil); oven temperature chromatographic column, evaluating the temperatures 39, 40 and 41 C; mobile phase flow rate between 0.99, 1 and 1.1 mL/min and
Drug Dev Ind Pharm, 2015; 41(1): 63–69
stability of the sample’s solution in 24 h of storage at 2–8 C. The linearity range of responses of the standards was determined on five concentration levels with three injections for each level. The parameter precision was evaluated on three levels; to evaluate the repeatability of the injection integration, the standard solutions and each sample were injected six times and the relative standard deviation values were calculated, while for intermediate precision test, the solutions were examined in triplicates for two consecutive days, with different analysts; the reproducibility was performed in two different laboratories, on the same day, in triplicate samples. The limits of detection (LOD) and limits of quantification (LOQ) were calculated by the ratio between the standard deviation of the linear coefficients of the three curves of the linearity test by averaging the slopes of the respective curves multiplied by 3 and 10, respectively. Accuracy was determined after the establishment of the linearity of the standards, being found from samples obtained from the raw material of BNZ in concentrations of 8, 12 and 16 mg/mL, in triplicate for each concentration, compared with the value obtained by same analysis performed on the standard of work. The specificity of the method was evaluated by: factor of resolution and relative retention time in existing construction of the curve with the linearity related substances (2-NIZ and NBCA). The selectivity was established by determining the purity of each peak using a DAD detector. Data were treated statistically by one way analysis of variance (ANOVA). A study of BNZ photodegradation in solid state by direct photolysis Samples of solid BNZ were exposed to an irradiation dose in a thin layer (100 mg) of about 1 mm in a petri dish for 41 h at 1 200 000 Lux and 200 W h/m2 in a photostability chamber6 (Nova EticaÕ , Vargem Grande Paulista, Brazil), calibrated using chemical and physical acthinometry with photon emission and UV radiation. The results were expressed as a percentage of BNZ and each analysis performed in triplicate, along with an HPLC – DAD assay, thermal analysis of BNZ by differential scanning calorimetry (DSC) and differential thermogravimetry analysis (DTG), mass spectrometry (MS), infrared spectrometry (IR). The results of the photostability experiment were assessed using Student’s t test, considering a significance level of 0.05. Thermal analysis of BNZ by differential scanning calorimetry and differential thermogravimetry DSC curves for the drug were obtained by means of a ShimadzuÕ (Japan) calorimeter, model DSC-60, in an atmosphere of nitrogen flow of 50 mL min1, with a sample mass of around 2.0 mg, packaged in aluminum crucibles, in order to heat them by 10 C min1 up to a temperature of 600 C. The calibration was performed in accordance with the DSC melting point of indium standard (156.6 0.3 C) and zinc (419.6 0.3 C). The heat flow and enthalpy were calibrated using the melting point of Indium (28.6 0.3 J g1) under the same conditions as the samples. Infrared spectroscopy The BNZ IR spectrum was obtained using a PerkinElmer SpectrumÕ 400 (Waltham, MA) infrared spectrometer, using an average of 10 sweeps from 4000 to 650 cm1 with a resolution of 4 cm1. Mass spectroscopy The samples were prepared in a similar manner to that described in the methodology used for analytical method indicative of
Photostability study of the Benznidazole
DOI: 10.3109/03639045.2013.845842
stability. The spectrometer used to analyze samples of BNZ was a Shimadzu thermal spray ionization and mass analyzer for time of flight, used for drug dilution in a acetonitrile-water (50:50, v/v) system, with an 80 to 300 m/z scan. The study of BNZ degradation by indirect photolysis
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The different physical mixtures (PM) of BNZ: excipient (1:1, w/w) were submitted to the stirrer Tubing Vortex, MarconiÕ brand, model MA 162. After shaking, the samples were placed in a photostability chamber at 25 C and the study was conducted using a method similar to the study of direct photolysis (the same methodology used for the study of BNZ photodegradation in solid state by direct photolysis) and later analyzed through the technique of DSC, according to methodology described above. The results were expressed as a percentage of BNZ and each analysis performed in triplicate, and an HPLC–DAD assay was carried out and evaluated using MS.
Results Study of direct photodegradation of BNZ In solid state, after exposure to 1 200 000 lux and 200 wh/m2 irradiation the BNZ was observed to change in color to a yellowish powder, demonstrating the degradation of the drug. The chromatograms of the solid samples showed that the BNZ is photosensitive, according to the ICH Standard 9, with a decay of 30% measured using HPLC after a degree of illumination/ irradiance determined by the regulatory bodies of 55% (Table 1). The development of stability indicating method was validated according to ICH standards. The summary results of method validation of BNZ are described in the Table 2. The rate of photochemical reactions may be controlled to a great extent by the crystalline. DSC technique can be used to monitor crystallinity loss of the drug after irradiation12. The DSC curve shown in Figure 1 reveals that the endothermic melting Table 1. Results of determination of BNZ content using HPLC and values of melting peak after submit PM irradiation. Determination of content using HPLC Sample BNZ BNZ photodegraded BNZ þ starch BNZ þ cellulose microcrystaline* BNZ þ croscarmellose BNZ þ colloidal silicon dioxide BNZ þ ethylcellulose BNZ þ estearate BNZ þ sodium starch glicolate BNZ þ lactose BNZ þ hpc BNZ þ hpmc BNZ þ pvp BNZ þ peg BNZ þ talc
%
T pico ( C)
98.7 67 63 57 71 75 72 79 70 71 67 64 93 79 64
191.9 189.4 188.8 187.8 188.4 187.5 187.6 187.3 186.6 188.0 187.6 189.1 – 185.3 188.7
*Critical excipient.
peak of BNZ of 191.1 C is reduced by 2 C after direct exposure to radiation. The exothermic peak of degradation of the drug, in turn, increases by about 4 C after exposure, from 287.5 C to 283.4 C. The energy required for fusion and degradation of the drug is markedly reduced, as confirmed by analysis of changes in enthalpy in the samples. These variations are predictable since the study of photodegradation of the drug caused decay of 30%, according to the HPLC results. Despite showing differences between the samples of drugs after direct radiation, the DSC curve is not selective for the identification of degradation products, because there was no observable melting peak characteristic of the new product formed during photolysis. The DTG curves for BNZ (Figure 2) show that, after radiation of the drug, the onset was anticipated around 13 C and shifted from 275.58 to 262.14 C. While the percentage of weight loss was 44.26% in BNZ, the photodegradated BNZ was reduced to 33.51%. This analysis, therefore, provides an effective visualization of the degradation of the drug. Nevertheless, no new mass loss events were observed in the chromatograms that might indicate the presence of degradation products. This technique, as well as the DSC technique, was not selective for the identification of photoproducts (PPs). Examination of the infrared spectra (IR) (Figure 3) showed that the sample of photodegraded BNZ was comparable to standard raw materials, showing discrete modifications in carbonyl regions, but is not conclusive to elucidation the chemical structure of BNZ degradations products. The chromatograms for the solid samples showed that the BNZ is photosensitive according to the standards of the ICH9. The degradation after the illumination/irradiation permitted by the regulatory bodies is 55%, compared to a degradation of BNZ (marked on light gray) of 30% here (Table 1), after undergoing the test conditions, resulting in two PPs, which are significant at the following retention times (Tr): PP-1 (Tr ¼ 2.75 min) and PP-2 (Tr ¼ 7.53 min) (Figure 4). Also on Table 1 are the PMs of BNZ plus excipients (marked on dark gray) that showed a significant decay. In order to shed light on the degradation route of BNZ, photoradiated samples of BNZ in isolation and in physical mixtures, the MS was used and showed a similar form of degradation of products in all samples. There are two possible ionizations – positive (Mþ) and negative (M) – assessed to establish how likely BNZ is to undergo them. The Figure 5 shows that positive ionization was much more effective in highlighting the real peak of most of the BNZ, with 100% showing the characteristic peak at 261.09 m/z, since the molecular mass of BNZ is 260 g/mol, so [M þ 1]þ ¼ 261.09 m/z, and it is also possible to observe [M þ 2]þ ¼ 262.09 m/z. In negative ionization, the main peaks, with intensity close to 100%, are in the region of 300 m/z, which is the characteristic peak of BNZ [M 1] ¼ 259.08 m/z with an intensity of approximately 50%. Therefore, analysis of the MS of the primary and secondary BNZ used those obtained by positive ionization, employing the negative ones only to confirm the results (Figure 5). The mass spectrum obtained from the two primary ionizations of BNZ is shown in Figure 5, showing that, in addition to the main
Table 2. Results of linearity, LOD and LOQ of method indicating stability of the BNZ. Substance 2-NIZ* BNZ NBCA*
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Equation of the line
R2
LOD (mg/mL)
LOQ (mg/mL)
y ¼ 253540,2 (8985,9) þ 29518,3 (182689,3) y ¼ 462405,4 (15383,0) þ 221556,3 (312746,3) y ¼ 576944,0 (11836,79) þ 187507,4 (240649,1)
0.9955 0.9961 0.9985
0.2613 0.03537 0.2058
0.8071 0.1179 0.6860
*2-NIZ e NBCA are two substances obtained from the photodegradation of BNZ.
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Drug Dev Ind Pharm, 2015; 41(1): 63–69
Figure 1. DSC curve for BNZ before and after radiation.
Figure 2. DTG curve of BNZ before and after radiation.
peak of BNZ [M þ 1], there are characteristic peaks at 139.99, 158.99, 176.99, 200.02 and 262.1. The peaks observed in the spectrum of BNZ represent secondary ionizations of the drug during the primary ionization or possible contaminants during sample preparation or degradation products produced during storage of the raw material. MS was conducted to obtain information on the secondary ionization of BNZ to confirm the degradation profile for thermal ionization of BNZ, with analysis by time of flight. By MS were observed only in the ionization of the peak corresponding to the BNZ, and these were of significant intensity, i.e. more than 10%, with five of 91.05, 107.04, 118.01, 157.07, and 214.09 m/z, which probably correspond to the chemical structures represented in Figure 6. The study of indirect photodegradation of BNZ
Figure 3. IR of standard BNZ (in gray) compared with photodegraded (black).
For the study of indirect photodegradation BNZ with 13 pharmaceutical excipients commonly used in the preparation of solid dosage forms were selected diluents, aggregating, disintegrants, lubricants and solubilizing agents. The results of the PM content and the value of the melting peak measured by DSC in BNZ PM submitted to the
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DOI: 10.3109/03639045.2013.845842
Photostability study of the Benznidazole
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Figure 4. Cromatogram of BNZ, BNZ photodegraded and BNZ þ cellulose photodegraded presenting two relative peaks PP-1 (Tr ¼ 2.75 min) and PP-2 (Tr ¼ 7.53 min).
Figure 5. BNZ mass spectra (MS) shows the positive ionization with characteristic peak at 261.09 m and negative ionization with characteristic peak at 259.08 m/z. Figure 6. Schema of secondary ionization of BNZ with probables corresponding of the chemical structures.
photostability chamber are shown in Table 1. Only the samples with PVP and PEG polymers were possible to observe variations in the apparent melting temperature of the drug. However, Lima3 reports the event to complete solubilization (if the PVP) or partial (with PEG) of the drug in the polymer before reaching the melting point of the BNZ, since these polymers melt before reaching 100 C. Among the other 11 PM there were no significant variations between the values of the peaks of drug in relation to
the melting point of the BNZ photodegraded (189.4 C). The DSC technique is not, therefore, a technique for assessing the differential thermal behavior of the BNZ in the presence of excipients. The HPLC technique showed more selective, since the DSC analyses are often a qualitative technique. Thus, for a quantitative assay of the BNZ’s PP, the HPLC analysis was the chosen one. To examine the BNZ content of the samples, the quantities of the drug measured in the samples were compared to
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Drug Dev Ind Pharm, 2015; 41(1): 63–69
Figure 7. Mass spectrum of BNZ, photodegraded BNZ and PM with photodegraded cellulose.
the internal and external working standard using the HPLC method described above. In this case the microcrystalline cellulose was a critical excipient because it had the lowest percentage of drug among the PM analyzed and the chromatogram for PM BNZ revealed degradation products with retention times almost overlapping those of the photoproducts (PP) found in the chromatogram of the drug without cellulose after photodegradation (Figure 4), with retention times of 2.5 min and 7.5 min, respectively. Therefore, this technique was chosen for determination of critical excipient that followed for analyses of mass spectra. The analysis of mass spectra, using methods described for analysis of indirect photodegradation, was also carried out for the sample and BNZ þ cellulose and confirms the result obtained using HPLC, because it is observed only an increase in intensity of the peaks corresponding to masses 184 and 79 g/mol, and also corresponding to the PPs found in the photodegradation of BNZ alone (Figure 7).
Figure 8. Possible degradation route of BNZ by photolysis.
Discussion In study of direct photodegradation of BNZ the chromatograms of the solid samples showed that the BNZ is photosensitive, according to the ICH standard9, with a decay of 30% measured using HPLC after a degree of illumination/irradiance determined by the regulatory bodies of 55%. These results are predictable in variations of DSC and DTG, but these techniques are not selective for the identification of degradation products. The exam of the infrared spectra (Figure 4) is not conclusive to elucidation the chemical structure of BNZ degradations products,
Photostability study of the Benznidazole
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DOI: 10.3109/03639045.2013.845842
thereby demonstrating that the degradation of BNZ was lower in the solid state, although it was not possible to visualize this without the isolation of the compounds. The MS used is the best equipment for the detection of degradation products of drugs because of its high resolution and high sensitivity, but these conditions are a disadvantage when looking at the amount of noise in the initial spectrum of the isolated molecule BNZ13. In order to shed light on the degradation route of BNZ, photoradiaded samples of BNZ in isolation and in physical mixtures were examined using a mass spectrometer, showing a similar form of degradation of products in all samples, and confirming the HPLC analysis that identified the formation of only two degradation products, shown in Figure 5. In addition, the chromatograph (HPLC-DAD), the mass spectrum evidence and theoretical data of a review of the literature were conducted on the mechanisms of photochemical degradation of molecules with functional groups similar to the BNZ. The chemical groups that link and free orbitals in valence band are more resistant to photolytic breakdown, owing to the fact that, when electrons are excited by photons of orbital change only, no energy absorption is required to break the dissipation that results in grouping7. In the case of double bonds between carbon and oxygen, both atoms (present in the amide group of the BNZ) have a free orbital in the last layer in this case, and this is also the valence: C6 ! 1s2 2s2 2p2/O8 ! 1s2 2s2 2p4. As a result, when these atoms are linked into double coordination (in orbitals), they form an electrostatic ‘‘barrier’’, which allows greater excitation of electrons, as they are not able to absorb the energy required for a homolytic breaking of the bounds7. This review revealed 3 photo-unstable points, which easily excited the BNZ molecule, and the carbons of the vicinal aromatic rings, thus producing the compounds depicted in Figure 8, where the first compound has a molecular mass of 183 g/mol and when positively ionized 184 m/z would be the second benzene ring. At study of indirect photodegradation the microcrystalline cellulose was the critical excipient in the study, because it had the lowest percentage of drug among the PM analyzed and is considered an ideal target for identifying the formation of degradation products not found in the study of direct photodegradation.
Conclusion The HPLC and MS techniques were used in identifying the degradation products of the BNZ after absorption of electromagnetic radiation from the photostability chamber used in this study. The chromatograms of the samples show two degradation products with retention times of 2.5 min and 7.5 min, as also observed in the spectrum (MS) with fragments with a load mass of 184 and 79 g/mol of nitro compounds and ring suggesting the proposed degradation route. The study of indirect photoreaction using the critical excipient cellulose microcrystalline showed there to be a lower content of BNZ after sample analysis, compared to irradiated BNZ, suggesting that the order of the process speeds up the degradation of the excipient. There was no
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new formation of degradation products in the chromatograms of BNZ mixtures with excipients (represented by cellulose microcrystalline), and the mass spectra of these samples are very similar to the spectrum of the drug after direct photolysis, which is only more intense at the peaks relating to the PPs.
Acknowledgements The authors Fabiana L. A. Santos, Larissa A. Rolim, Camila B. M. Figueireˆdo, Magaly A. M. Lyra, S. Monize Peixoto and Leslie R. M. Ferraz had substantial contributions to conception and design, acquisition and interpretation of data, and preparation of the article. Jose´ Lamartine Soares-Sobrinho, Adley Antonini Neves de Lima and Ana Cristina Leite Lima participated and adviced critically important intellectual content. Pedro Jose´ Rolim Neto approved the final version to be published.
Declaration of interest The authors report no declarations of interest.
References 1. Drugs for Neglected Diseases Initiative, DNDi. Doenc¸a de Chagas. Available from: http://www.dndi.org.br/pt/doencas-negligenciadas/ doenca-de-chagas.htm [last accessed 6 Jan 2013]. 2. Leite ACL, Donnici CL, Arau´jo MH, et al. Ruthenium complexes endowed with potent anti-Trypanosoma cruzi activity: synthesis, biological characterization and structure–activity relationships. Bioorg Med Chem 2009;17:5038–43. 3. Lima AAN, Soares-Sobrinho JL, Silva JL, et al. The use of solid dispersion systems in hydrophilic carriers to increase Benznidazole solubility. J Pharm Sci 2011;100:2443–51. 4. Soares-Sobrinho JL, Soares MFR, Lopes PQ, et al. A preformulation study of a New Medicine for Chagas disease treatment: physicochemical characterization, thermal stability, and compatibility of Benznidazole. AAPS Pharm Sci Tech 2010;11:1391–6. 5. Silva KER, Alves LDS, Soares MFR, et al. Modelos de Avaliac¸a˜o da Estabilidade de Fa´rmacos e Medicamentos para a Indu´stria Farmaceˆutica. Rev Cieˆnc Farm Ba´sica Apl 2009;30:1–8. 6. Piechock JT, Thoma K. Pharmaceutical photostability and stabilization technology. New York: Informa Healthcare; 2007. 7. Tonnesen HH. Photostability of drugs and drug formulations. 2nd ed. London: CRC Press; 2004. 8. Kuhn HJ, Braslavsky SE, Schimidt R. Chemical actinometry. Pure Appl Chem 2009;76:2105–46. 9. ICH. Stability Testing: Photostability Testing of New Drugs substances and Products Q1B, 2003. Available from: http:// www.ich.org [last accessed 15 March 2013]. 10. Silva ALM, Soares-Sobrinho JL, Rolim-Neto PJ, et al. Desenvolvimento de Me´todo Analı´tico por CLAE em Comprimidos de Benznidazol para a doenc¸a de Chagas. Quı´m Nova 2007;30:1163–6. 11. International Conference on Harmonization, ‘‘ICH Q2B: Validation of Analytical Procedures: Terms and Definitions,’’ Step 5 (1996). Avaiable from: http://www.ich.org/products/guidelines/quality/article/quality-guidelines.html [last accessed 2 January 2013]. 12. Ramadan A, El-Massik M, El-Khordagui L, et al. Surface treatment: a potential approach for enhancement of solid-state photostability. Int J Pharm 2006;307:141–9. 13. Silverstein RM, Webster FX, Kiemle DJ. Identificac¸a˜o Espectrome´trica de Compostos Orgaˆnicos. 7th ed. Rio de Janeiro: Livros Te´cnicos e Cientı´ficos Editora S.A.; 2007.
RESEARCH ARTICLE
A study of photostability and compatibility of the anti-chagas drug Benznidazole with pharmaceutics excipients
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Fabiana L. A. Santos1, Larissa A. Rolim1, Camila B. M. Figueireˆdo1, Magaly A. M. Lyra1, Monize S. Peixoto1, Leslie R. M. Ferraz1, Jose´ L. Soares-Sobrinho1, A´dley Antonini Neves de Lima1,2, Ana Cristina Lima Leite3, and Pedro Jose´ Rolim Neto1 1
Laborato´rio de Tecnologia dos Medicamentos, Departamento de Cieˆncias Farmaceˆuticas, Universidade Federal de Pernambuco, Rua Prof. Arthur de Sa´, s/n, Cidade Universita´ria, Recife, PE, Brazil, 2Laborato´rio de Tecnologia Farmaceˆutica, Faculdade de Cieˆncias Farmaceˆuticas, Universidade Federal do Amazonas, Rua Alexandre Amorim, no. 330, Aparecida, Manaus, AM, Brazil, and 3Laborato´rio de Planejamento, Avaliac¸a˜o e Sı´ntese de Fa´rmacos, Departamento de Cieˆncias Farmaceˆuticas, Universidade Federal de Pernambuco, Rua Prof. Arthur de Sa´, s/n, Cidade Universita´ria, Recife, PE, Brazil Abstract
Keywords
Context: Benznidazole (BNZ) is an antiparasitic with trypanocidal properties for the etiological treatment of Chagas disease since 1973. Monitoring the stability of this drug is one of the most effective methods of assessment, forecasting and prevention of problems related to quality product. Objective: To investigate the direct and indirect photodegradation of BNZ and to evaluate the interference of the excipients used in the forms dosage solid as well as to shed light on the chemical structure of the degradation products obtained. Materials and methods: To perform this work we adopted the ‘‘ICH Harmonised Tripartite Guideline: Photostability Testing of New Drug Substances and Products Q1B’’ (Guideline Q1B). We used benzonidazole (BNZ) (N-benzil-2-(2-nitroimidazol-1-il) acetamide) (LAFEPEÕ , Recife, Brazil) and various excipients; beyond high-performance liquid chromatography (HPLC), differential scanning calorimetry (DSC), infrared spectroscopy (IR) and mass spectrometry/mass spectrometry (MS/MS). The indirect photodegradation of BNZ was carried out using physical mixtures with 13 pharmaceutical excipients commonly used in the preparation of solid dosage forms. Results: HPLC and MS/MS techniques were selected for the identification of two photoproducts (PPs) and photoreactions found in direct and indirect tests with the microcrystalline cellulose, considered a critical excipient. Discussion: Despite variations in the infrared spectrometry, differential scanning calorimetry and differential thermogravimetry curves, these techniques are not conclusive since the study of photodegradation of the drug caused decay of 30%, according to the ICH. Conclusions: The results show that BNZ only undergoes direct photodegradation, since no new PPs were found for a combination of the drug and excipients.
Benznidazole, Chagas disease, degradation product, HPLC-MS/MS, photodegradation
Introduction Endemic in 21 Latin American countries, Chagas disease kills every year, more people in the region than any other parasitic disease, including malaria1. Benznidazole (N-benzyl-2-nitro-1imidazolacetamide) is an antiparasitic, nitroimidazole derivative with trypanocidal properties, which has been used since 1973 for the etiological treatment of Chagas disease2. Two of the principal problems with this drug are its toxicity and low solubility3.
Address for correspondence: Fabiana Lı´cia Arau´jo dos Santos, Laborato´rio de Tecnologia dos Medicamentos, Departamento de Cieˆncias Farmaceˆuticas, Universidade de Pernambuco. Rua Professor Arthur de Sa´, s/n, Cidade Universita´ria, 50740-521 Recife, PE, Brasil. Tel: +55(81) 3272-1383. E-mail: [email protected]
History Received 11 July 2013 Revised 6 September 2013 Accepted 9 September 2013 Published online 17 October 2013
Despite the importance of treatment of the disease Benznidazole, few studies discuss the stability of the drug in the presence of light. In 2010, Soares-Sobrinho4 conducted a compatibility study during a tablet preformulation study and compared with the commercially available tablet, but he did not assess the formation of photodegradation products. Monitoring the stability of drugs is one of the most effective methods of assessment, forecasting and prevention of problems related to quality product during its validity. The safety and efficacy can also be evaluated by monitoring the formation of degradation products that may cause loss of therapeutic activity or toxicity5. In a situation where the isolated drugs absorb ultraviolet or visible radiation and this results in the loss of the drug and the formation of degradation products, there is a direct photoreaction. In the case of indirect photoreactions, it is not the drug, but another component of the formulation (e.g. excipients or impurities) that
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absorb ultraviolet or visible radiation, resulting in the formation of one or more reactive species which react with the drugs6–8. Thus, the aim of this study was to investigate the direct and indirect photodegradation of BNZ in solid state and to shed light on the chemical structure of the degradation products obtained, to evaluate the interference of the excipients used in the formulation commercially available and other excipients which may be used to obtain pharmaceutical forms that can solve the problems of solubility of the drug. To perform this work we adopted the ‘‘ICH Harmonised Tripartite Guideline: Photostability Testing of New Drug Susbtances and Products Q1B’’ (Guideline Q1B)9.
Material and methods
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Materials BNZ 99.9% (purity determined by high performance liquid chromatography (HPLC), using methods developed by Silva in 2007)10, N-benzil-2-(2-nitroimidazol-1-il)acetamide, raw material, and the standard were provided by Laborato´rio Farmaceˆutico do Estado de Pernambuco (LAFEPEÕ , Recife, Brazil), donated by RocheÕ Pharmaceutical Industry. All solvents and other substances used were of analytical grade. The solutions were prepared using ultrapure water (Milli QÕ , Billerica, MA) and filtered through 0.22 mm porosity filter membranes (MilliporeÕ , Billerica, MA). The excipients used were from various commercial sources: cellulose microcristaline, croscarmellose sodium, sodium starch glycolate and magnesium stearate (Blanver and MicrocelÕ Ltd, Itapevi, Brazil), starch 1500Õ and hydroxypropylmetilcellulose (HPMC) (ColorconÕ , West Point, MS), lactose (MeggleÕ , Wasserburg, Germany), talc (MagnesitaÕ , Contagem, Brazil), hydroxypropylcellulose (HPC) (Aqualon, Wilmington, DE), ethylcellulose, polyethylenoglycol (PEG 4000), polyvinylpirrolidone – PVP K30 (ISP CorpÕ , Miami, FL) and colloidal silicon dioxide (HenkelÕ , Du¨sseldorf, Brazil). Analytical method indicative of stability The BNZ assay was performed using HPLC on a ShimadzuÕ (Kyoto, Japan) with a controller system (Shimadzu SCL – 20VP), pumps A and B (Shimadzu LC – 20AD VP), a PDA detector (SPDM20A VP), auto-injector (Shimadzu SIL – 20AD VP), and oven (Shimadzu CTO – 20A) and the chromatograms were drawn up using the LC program – SolutionÕ 1.0. The following analytical conditions were used: column reverse phase ShimadzuÕ , C18, 5 mm, 25 cm 0.46 cm, flow 1.0 mL min1, injection volume: 40 mL, a wavelength of 210 nm, temperature 40 C and mobile phase: acetonitrile – phosphate buffer (pH 2.7; 20 mM) (80:20, v/v). Validation analytical method In the process of validation of analytical methods developed were assessed the following parameters: robustness, linearity, range, detection limit, quantification limit, precision, accuracy and selectivity11. Each sample, followed by a standard curve, was prepared from a stock solution daily, in concentrations of 8, 12 and 16 mg/mL containing the BNZ and their chemical reference substances (CRS): sulfate aminoimidazol, 2-nitroimidazole (2-NIZ) and N-benzilcloroacetamida (NBCA) simultaneously. In the robustness test variables were analyzed: the manufacturer of the dibasic potassium phosphate (mobile phase component), evaluating the following marks: Carlo ErbaÕ (Rodano, Italy) and SinthÕ (Diadema, Brazil); oven temperature chromatographic column, evaluating the temperatures 39, 40 and 41 C; mobile phase flow rate between 0.99, 1 and 1.1 mL/min and
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stability of the sample’s solution in 24 h of storage at 2–8 C. The linearity range of responses of the standards was determined on five concentration levels with three injections for each level. The parameter precision was evaluated on three levels; to evaluate the repeatability of the injection integration, the standard solutions and each sample were injected six times and the relative standard deviation values were calculated, while for intermediate precision test, the solutions were examined in triplicates for two consecutive days, with different analysts; the reproducibility was performed in two different laboratories, on the same day, in triplicate samples. The limits of detection (LOD) and limits of quantification (LOQ) were calculated by the ratio between the standard deviation of the linear coefficients of the three curves of the linearity test by averaging the slopes of the respective curves multiplied by 3 and 10, respectively. Accuracy was determined after the establishment of the linearity of the standards, being found from samples obtained from the raw material of BNZ in concentrations of 8, 12 and 16 mg/mL, in triplicate for each concentration, compared with the value obtained by same analysis performed on the standard of work. The specificity of the method was evaluated by: factor of resolution and relative retention time in existing construction of the curve with the linearity related substances (2-NIZ and NBCA). The selectivity was established by determining the purity of each peak using a DAD detector. Data were treated statistically by one way analysis of variance (ANOVA). A study of BNZ photodegradation in solid state by direct photolysis Samples of solid BNZ were exposed to an irradiation dose in a thin layer (100 mg) of about 1 mm in a petri dish for 41 h at 1 200 000 Lux and 200 W h/m2 in a photostability chamber6 (Nova EticaÕ , Vargem Grande Paulista, Brazil), calibrated using chemical and physical acthinometry with photon emission and UV radiation. The results were expressed as a percentage of BNZ and each analysis performed in triplicate, along with an HPLC – DAD assay, thermal analysis of BNZ by differential scanning calorimetry (DSC) and differential thermogravimetry analysis (DTG), mass spectrometry (MS), infrared spectrometry (IR). The results of the photostability experiment were assessed using Student’s t test, considering a significance level of 0.05. Thermal analysis of BNZ by differential scanning calorimetry and differential thermogravimetry DSC curves for the drug were obtained by means of a ShimadzuÕ (Japan) calorimeter, model DSC-60, in an atmosphere of nitrogen flow of 50 mL min1, with a sample mass of around 2.0 mg, packaged in aluminum crucibles, in order to heat them by 10 C min1 up to a temperature of 600 C. The calibration was performed in accordance with the DSC melting point of indium standard (156.6 0.3 C) and zinc (419.6 0.3 C). The heat flow and enthalpy were calibrated using the melting point of Indium (28.6 0.3 J g1) under the same conditions as the samples. Infrared spectroscopy The BNZ IR spectrum was obtained using a PerkinElmer SpectrumÕ 400 (Waltham, MA) infrared spectrometer, using an average of 10 sweeps from 4000 to 650 cm1 with a resolution of 4 cm1. Mass spectroscopy The samples were prepared in a similar manner to that described in the methodology used for analytical method indicative of
Photostability study of the Benznidazole
DOI: 10.3109/03639045.2013.845842
stability. The spectrometer used to analyze samples of BNZ was a Shimadzu thermal spray ionization and mass analyzer for time of flight, used for drug dilution in a acetonitrile-water (50:50, v/v) system, with an 80 to 300 m/z scan. The study of BNZ degradation by indirect photolysis
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The different physical mixtures (PM) of BNZ: excipient (1:1, w/w) were submitted to the stirrer Tubing Vortex, MarconiÕ brand, model MA 162. After shaking, the samples were placed in a photostability chamber at 25 C and the study was conducted using a method similar to the study of direct photolysis (the same methodology used for the study of BNZ photodegradation in solid state by direct photolysis) and later analyzed through the technique of DSC, according to methodology described above. The results were expressed as a percentage of BNZ and each analysis performed in triplicate, and an HPLC–DAD assay was carried out and evaluated using MS.
Results Study of direct photodegradation of BNZ In solid state, after exposure to 1 200 000 lux and 200 wh/m2 irradiation the BNZ was observed to change in color to a yellowish powder, demonstrating the degradation of the drug. The chromatograms of the solid samples showed that the BNZ is photosensitive, according to the ICH Standard 9, with a decay of 30% measured using HPLC after a degree of illumination/ irradiance determined by the regulatory bodies of 55% (Table 1). The development of stability indicating method was validated according to ICH standards. The summary results of method validation of BNZ are described in the Table 2. The rate of photochemical reactions may be controlled to a great extent by the crystalline. DSC technique can be used to monitor crystallinity loss of the drug after irradiation12. The DSC curve shown in Figure 1 reveals that the endothermic melting Table 1. Results of determination of BNZ content using HPLC and values of melting peak after submit PM irradiation. Determination of content using HPLC Sample BNZ BNZ photodegraded BNZ þ starch BNZ þ cellulose microcrystaline* BNZ þ croscarmellose BNZ þ colloidal silicon dioxide BNZ þ ethylcellulose BNZ þ estearate BNZ þ sodium starch glicolate BNZ þ lactose BNZ þ hpc BNZ þ hpmc BNZ þ pvp BNZ þ peg BNZ þ talc
%
T pico ( C)
98.7 67 63 57 71 75 72 79 70 71 67 64 93 79 64
191.9 189.4 188.8 187.8 188.4 187.5 187.6 187.3 186.6 188.0 187.6 189.1 – 185.3 188.7
*Critical excipient.
peak of BNZ of 191.1 C is reduced by 2 C after direct exposure to radiation. The exothermic peak of degradation of the drug, in turn, increases by about 4 C after exposure, from 287.5 C to 283.4 C. The energy required for fusion and degradation of the drug is markedly reduced, as confirmed by analysis of changes in enthalpy in the samples. These variations are predictable since the study of photodegradation of the drug caused decay of 30%, according to the HPLC results. Despite showing differences between the samples of drugs after direct radiation, the DSC curve is not selective for the identification of degradation products, because there was no observable melting peak characteristic of the new product formed during photolysis. The DTG curves for BNZ (Figure 2) show that, after radiation of the drug, the onset was anticipated around 13 C and shifted from 275.58 to 262.14 C. While the percentage of weight loss was 44.26% in BNZ, the photodegradated BNZ was reduced to 33.51%. This analysis, therefore, provides an effective visualization of the degradation of the drug. Nevertheless, no new mass loss events were observed in the chromatograms that might indicate the presence of degradation products. This technique, as well as the DSC technique, was not selective for the identification of photoproducts (PPs). Examination of the infrared spectra (IR) (Figure 3) showed that the sample of photodegraded BNZ was comparable to standard raw materials, showing discrete modifications in carbonyl regions, but is not conclusive to elucidation the chemical structure of BNZ degradations products. The chromatograms for the solid samples showed that the BNZ is photosensitive according to the standards of the ICH9. The degradation after the illumination/irradiation permitted by the regulatory bodies is 55%, compared to a degradation of BNZ (marked on light gray) of 30% here (Table 1), after undergoing the test conditions, resulting in two PPs, which are significant at the following retention times (Tr): PP-1 (Tr ¼ 2.75 min) and PP-2 (Tr ¼ 7.53 min) (Figure 4). Also on Table 1 are the PMs of BNZ plus excipients (marked on dark gray) that showed a significant decay. In order to shed light on the degradation route of BNZ, photoradiated samples of BNZ in isolation and in physical mixtures, the MS was used and showed a similar form of degradation of products in all samples. There are two possible ionizations – positive (Mþ) and negative (M) – assessed to establish how likely BNZ is to undergo them. The Figure 5 shows that positive ionization was much more effective in highlighting the real peak of most of the BNZ, with 100% showing the characteristic peak at 261.09 m/z, since the molecular mass of BNZ is 260 g/mol, so [M þ 1]þ ¼ 261.09 m/z, and it is also possible to observe [M þ 2]þ ¼ 262.09 m/z. In negative ionization, the main peaks, with intensity close to 100%, are in the region of 300 m/z, which is the characteristic peak of BNZ [M 1] ¼ 259.08 m/z with an intensity of approximately 50%. Therefore, analysis of the MS of the primary and secondary BNZ used those obtained by positive ionization, employing the negative ones only to confirm the results (Figure 5). The mass spectrum obtained from the two primary ionizations of BNZ is shown in Figure 5, showing that, in addition to the main
Table 2. Results of linearity, LOD and LOQ of method indicating stability of the BNZ. Substance 2-NIZ* BNZ NBCA*
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Equation of the line
R2
LOD (mg/mL)
LOQ (mg/mL)
y ¼ 253540,2 (8985,9) þ 29518,3 (182689,3) y ¼ 462405,4 (15383,0) þ 221556,3 (312746,3) y ¼ 576944,0 (11836,79) þ 187507,4 (240649,1)
0.9955 0.9961 0.9985
0.2613 0.03537 0.2058
0.8071 0.1179 0.6860
*2-NIZ e NBCA are two substances obtained from the photodegradation of BNZ.
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Figure 1. DSC curve for BNZ before and after radiation.
Figure 2. DTG curve of BNZ before and after radiation.
peak of BNZ [M þ 1], there are characteristic peaks at 139.99, 158.99, 176.99, 200.02 and 262.1. The peaks observed in the spectrum of BNZ represent secondary ionizations of the drug during the primary ionization or possible contaminants during sample preparation or degradation products produced during storage of the raw material. MS was conducted to obtain information on the secondary ionization of BNZ to confirm the degradation profile for thermal ionization of BNZ, with analysis by time of flight. By MS were observed only in the ionization of the peak corresponding to the BNZ, and these were of significant intensity, i.e. more than 10%, with five of 91.05, 107.04, 118.01, 157.07, and 214.09 m/z, which probably correspond to the chemical structures represented in Figure 6. The study of indirect photodegradation of BNZ
Figure 3. IR of standard BNZ (in gray) compared with photodegraded (black).
For the study of indirect photodegradation BNZ with 13 pharmaceutical excipients commonly used in the preparation of solid dosage forms were selected diluents, aggregating, disintegrants, lubricants and solubilizing agents. The results of the PM content and the value of the melting peak measured by DSC in BNZ PM submitted to the
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DOI: 10.3109/03639045.2013.845842
Photostability study of the Benznidazole
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Figure 4. Cromatogram of BNZ, BNZ photodegraded and BNZ þ cellulose photodegraded presenting two relative peaks PP-1 (Tr ¼ 2.75 min) and PP-2 (Tr ¼ 7.53 min).
Figure 5. BNZ mass spectra (MS) shows the positive ionization with characteristic peak at 261.09 m and negative ionization with characteristic peak at 259.08 m/z. Figure 6. Schema of secondary ionization of BNZ with probables corresponding of the chemical structures.
photostability chamber are shown in Table 1. Only the samples with PVP and PEG polymers were possible to observe variations in the apparent melting temperature of the drug. However, Lima3 reports the event to complete solubilization (if the PVP) or partial (with PEG) of the drug in the polymer before reaching the melting point of the BNZ, since these polymers melt before reaching 100 C. Among the other 11 PM there were no significant variations between the values of the peaks of drug in relation to
the melting point of the BNZ photodegraded (189.4 C). The DSC technique is not, therefore, a technique for assessing the differential thermal behavior of the BNZ in the presence of excipients. The HPLC technique showed more selective, since the DSC analyses are often a qualitative technique. Thus, for a quantitative assay of the BNZ’s PP, the HPLC analysis was the chosen one. To examine the BNZ content of the samples, the quantities of the drug measured in the samples were compared to
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Figure 7. Mass spectrum of BNZ, photodegraded BNZ and PM with photodegraded cellulose.
the internal and external working standard using the HPLC method described above. In this case the microcrystalline cellulose was a critical excipient because it had the lowest percentage of drug among the PM analyzed and the chromatogram for PM BNZ revealed degradation products with retention times almost overlapping those of the photoproducts (PP) found in the chromatogram of the drug without cellulose after photodegradation (Figure 4), with retention times of 2.5 min and 7.5 min, respectively. Therefore, this technique was chosen for determination of critical excipient that followed for analyses of mass spectra. The analysis of mass spectra, using methods described for analysis of indirect photodegradation, was also carried out for the sample and BNZ þ cellulose and confirms the result obtained using HPLC, because it is observed only an increase in intensity of the peaks corresponding to masses 184 and 79 g/mol, and also corresponding to the PPs found in the photodegradation of BNZ alone (Figure 7).
Figure 8. Possible degradation route of BNZ by photolysis.
Discussion In study of direct photodegradation of BNZ the chromatograms of the solid samples showed that the BNZ is photosensitive, according to the ICH standard9, with a decay of 30% measured using HPLC after a degree of illumination/irradiance determined by the regulatory bodies of 55%. These results are predictable in variations of DSC and DTG, but these techniques are not selective for the identification of degradation products. The exam of the infrared spectra (Figure 4) is not conclusive to elucidation the chemical structure of BNZ degradations products,
Photostability study of the Benznidazole
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DOI: 10.3109/03639045.2013.845842
thereby demonstrating that the degradation of BNZ was lower in the solid state, although it was not possible to visualize this without the isolation of the compounds. The MS used is the best equipment for the detection of degradation products of drugs because of its high resolution and high sensitivity, but these conditions are a disadvantage when looking at the amount of noise in the initial spectrum of the isolated molecule BNZ13. In order to shed light on the degradation route of BNZ, photoradiaded samples of BNZ in isolation and in physical mixtures were examined using a mass spectrometer, showing a similar form of degradation of products in all samples, and confirming the HPLC analysis that identified the formation of only two degradation products, shown in Figure 5. In addition, the chromatograph (HPLC-DAD), the mass spectrum evidence and theoretical data of a review of the literature were conducted on the mechanisms of photochemical degradation of molecules with functional groups similar to the BNZ. The chemical groups that link and free orbitals in valence band are more resistant to photolytic breakdown, owing to the fact that, when electrons are excited by photons of orbital change only, no energy absorption is required to break the dissipation that results in grouping7. In the case of double bonds between carbon and oxygen, both atoms (present in the amide group of the BNZ) have a free orbital in the last layer in this case, and this is also the valence: C6 ! 1s2 2s2 2p2/O8 ! 1s2 2s2 2p4. As a result, when these atoms are linked into double coordination (in orbitals), they form an electrostatic ‘‘barrier’’, which allows greater excitation of electrons, as they are not able to absorb the energy required for a homolytic breaking of the bounds7. This review revealed 3 photo-unstable points, which easily excited the BNZ molecule, and the carbons of the vicinal aromatic rings, thus producing the compounds depicted in Figure 8, where the first compound has a molecular mass of 183 g/mol and when positively ionized 184 m/z would be the second benzene ring. At study of indirect photodegradation the microcrystalline cellulose was the critical excipient in the study, because it had the lowest percentage of drug among the PM analyzed and is considered an ideal target for identifying the formation of degradation products not found in the study of direct photodegradation.
Conclusion The HPLC and MS techniques were used in identifying the degradation products of the BNZ after absorption of electromagnetic radiation from the photostability chamber used in this study. The chromatograms of the samples show two degradation products with retention times of 2.5 min and 7.5 min, as also observed in the spectrum (MS) with fragments with a load mass of 184 and 79 g/mol of nitro compounds and ring suggesting the proposed degradation route. The study of indirect photoreaction using the critical excipient cellulose microcrystalline showed there to be a lower content of BNZ after sample analysis, compared to irradiated BNZ, suggesting that the order of the process speeds up the degradation of the excipient. There was no
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new formation of degradation products in the chromatograms of BNZ mixtures with excipients (represented by cellulose microcrystalline), and the mass spectra of these samples are very similar to the spectrum of the drug after direct photolysis, which is only more intense at the peaks relating to the PPs.
Acknowledgements The authors Fabiana L. A. Santos, Larissa A. Rolim, Camila B. M. Figueireˆdo, Magaly A. M. Lyra, S. Monize Peixoto and Leslie R. M. Ferraz had substantial contributions to conception and design, acquisition and interpretation of data, and preparation of the article. Jose´ Lamartine Soares-Sobrinho, Adley Antonini Neves de Lima and Ana Cristina Leite Lima participated and adviced critically important intellectual content. Pedro Jose´ Rolim Neto approved the final version to be published.
Declaration of interest The authors report no declarations of interest.
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