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ESI-Q-TOF
Journal of Pharmaceutical and Biomedical Analysis 131 (2016) 272–280
Contents lists available at ScienceDirect
Journal of Pharmaceutical and Biomedical Analysis journal homepage: www.elsevier.com/locate/jpba
Characterization of forced degradation products of clozapine by LC-DAD/ESI-Q-TOF ∗ ´ ´ Robert Skibinski , Jakub Trawinski, Łukasz Komsta, Katarzyna Bajda Department of Medicinal Chemistry, Medical University of Lublin, Jaczewskiego 4, 20-090 Lublin, Poland
a r t i c l e
i n f o
Article history: Received 28 June 2016 Received in revised form 29 August 2016 Accepted 3 September 2016 Available online 4 September 2016 Keywords: Mass spectrometry UHPLC Stressed degradation Photodegradation Clozapine
a b s t r a c t Forced degradation of clozapine in solutions under acidic, basic, neutral, photo UV–vis, photo UVC and oxidative stress conditions was investigated and structural elucidation of its degradation products was performed with the use of the UHPLC-DAD system coupled with accurate hybrid ESI-Q-TOF mass spectrometer. The developed method allows to collect all essential data for the determination of degradation kinetics and for the structural elucidation of the formed products. Six degradation products were found and their masses and formulas were obtained with high accuracy (0.61–3.75 ppm). For all the analyzed compounds MS/MS fragmentation spectra were also obtained allowing structural elucidation of the unknown degradation products. It was found that the decomposition of clozapine yields the first-order kinetic reaction in all stress conditions and it is fragile towards acidic hydrolysis and oxidative conditions. Additionally, PCA analysis of registered TOF (MS) forced degradation profiles of clozapine shows no differences between the samples obtained from pharmaceutical formulation and bulk substance. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Clozapine (8-chloro-11-(4-methylpiperazin-1-yl)-5Hdibenzo[b,e][1,4]diazepine) is the first atypical antipsychotic medication introduced to the market in 1960s as an effective agent to schizophrenia treatment [1]. Its pharmacological activity is based on the selective 5-HT2A and D4 receptor antagonist together with its weak D2 receptor blocking [2]. Clozapine is characterized by superior efficacy in relieving the positive and negative symptoms in treatment-resistant schizophrenic patients with minimal extrapyramidal side effects, however with the risk of agranulocytosis [3,4]. Stress testing studies are an integral part of the stability testing of drugs and must be considered during the development and registration process of pharmaceuticals. The degradation of drugs is an important part of investigation because this process can result in the loss of drug effectiveness as well as it can lead to additional adverse effects due to the formation of toxic degradation products. From this point of view it is very important to know what products (impurities) are formed from the medicines during the degradation process and what their exact chemical structure is. This information can be very useful for the manufacturing, quality control, storage and administration of pharmaceuticals [5–7].
∗ Corresponding author. ´ E-mail address: [email protected] (R. Skibinski). http://dx.doi.org/10.1016/j.jpba.2016.09.007 0731-7085/© 2016 Elsevier B.V. All rights reserved.
The degradation and stability study of clozapine was not studied exactly so far and only some papers concerning the development of stability-indicating method for the analysis of this drug with the use of TLC [8,9], LC-UV and UV–vis spectrophotometry [9] are published. In these papers no structural elucidation of degradation products was performed and only Hasan et al. [9] noticed that under acidic hydrolysis one degradation product − N-methylpiperazine was obtained. The stability of clozapine was also investigated in oral suspension at room temperature by the use of LC-UV method [10]. Hence, it is necessary to carry out the forced degradation study of clozapine including the structure elucidation of the formed products. For this purpose a new analytical method with the use of UHPLC-DAD system coupled with accurate hybrid ESI-Q-TOF mass spectrometer was developed. Additionally, the multivariate chemometric analysis (PCA) of forced degradation profiles of clozapine from bulk substance and pharmaceutical formulation was performed.
2. Experimental 2.1. Chemicals and reagents Clozapine and water for LC–MS Ultra grade were obtained from Sigma-Aldrich (St. Louis, USA). Methanol hypergrade for LC–MS and acetonitrile gradient grade were purchased from Merck (Darmstadt, Germany) and 98% formic acid for mass spectroscopy was
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obtained from Fluka (Taufkirchen, Germany). All analytical grade reagents (hydrochloric acid, sodium hydroxide, 30% hydrogen peroxide) were purchased from POCh (Gliwice, Poland). Klozapol tablets (100 mg of clozapine) were obtained from Anpharm (Warsaw, Poland). 2.2. LC-DAD/ESI-Q-TOF analysis LC–MS analysis was performed with the use of Agilent AccurateMass Q-TOF LC/MS G6520 B system with dual electrospray (DESI) ionization source and Infinity 1290 ultra-high-pressure liquid chromatography system consisting of: binary pomp G4220A, FC/ALS thermostat G1330B, autosampler G4226A, DAD detector G4212A, TCC G1316C module and Zorbax Eclipse-C18 (2.1 × 50 mm, dp = 1.8 m) HD column (Agilent Technologies, Santa Clara, USA). A mixture of methanol (A) and water (B) with addition of 0.1% solution of formic acid in both media was used as a mobile phase. The gradient elution was carried out at constant flow 0.3 ml/min from 20%A (80%B) 0 − 1 min and then 20%A to 70%A 1–7.5 min 1 min post time was performed to return to initial conditions. The injection volume was 1 l and the column temperature was maintained at 35 ◦ C. MassHunter workstation software in version B.04.00 was used for the control of the system, data acquisition, qualitative and quantitative analysis. The optimization of the instrument conditions was set out from the proper tuning of Q-TOF detector in a positive mode with the use of Agilent ESI-L tuning mix in the extended dynamic range (2 GHz). The following instrument settings were applied: gas temp.: 300 ◦ C, drying gas: 10 l/min, nebulizer pressure: 40 psig, capillary voltage: 3000 V, fragmentor voltage: 110 V, skimmer voltage: 65 V, octopole 1 RF voltage: 250 V. Data acquisition was performed in centroids with the use of TOF (MS) and also targeted MS/MS mode. The spectral parameters for both modes were: mass range: 50–950 m/z and the acquisition rate: 1.2 spectra/s. The collision energy (CID) for MS/MS experiments was ranged: 7.8–25.7 eV. To ensure accuracy in masses measurements, a reference mass correction was used and masses 121.050873 and 922.009798 were used as lock masses. DAD detector (200–400 nm) was also used for the additional monitoring and quantitation analysis of clozapine ( = 290 nm).
Fig. 1. The 3D PCA plot of forced degradation profiles of standard (squares) and pharmaceutical formulation (triangles) samples of clozapine.
2.3. Forced degradation studies Forced degradation studies were performed independently for the bulk substance (STD) and pharmaceutical formulation (TAB) of clozapine. Stock STD and TAB solutions of clozapine were prepared in acetonitrile at concentration 500 g mL−1 . In the case of TAB solution the equivalent of 12,5 mg of clozapine from mortared 20 Klozapol tablets was transferred to 25 ml volumetric flask and after the addition of acetonitrile extracted by shaker. The obtained suspension was centrifuged and next used as a stock solution. The working solutions were prepared by diluting stock solutions using the proper solvent to obtain a final concentration of 10 g mL−1 and next stressed under hydrolytic, oxidative and photolytic conditions (Table 1). All the hydrolytic and oxidative tests were performed using 10 ml of working solution placed in hermetically sealed glass vials. For the photodegradation tests the working solutions were placed in a quartz caped cells (l = 1 cm) mounted horizontally and irradiated with UV–vis or UVC radiation. The distance between the lamp and the samples was 10 cm in both cases. As a UV–vis source a photostability chamber Atlas Suntest CPS+ (Linsengericht, Germany) with full UV–vis spectrum (ID65) according to ICH guidelines was used. The irradiance was set to 250 W/m2 which corresponds to the dose of 900 kJ/m2 /h. Under these conditions the recommended ICH dose (1200000 lx of light intensity) was
Fig. 2. TOF total ion chromatograms obtained under acidic (A), basic (B), neutral (C), photo UV–vis (D), photo UVC (E) and oxidative (F) stress conditions.
reached after 21 h of irradiation. As a UVC source Haland HA-05 (Warsaw, Poland) ultraviolet laboratory lamp equipped with 6W quartz ultraviolet tube emitting mercury spectrum with 254 nm principal line was used. The average UVC irradiation intensity was 7.5 W/m2 . The dark control samples were also performed for both photostability experiments by exposing the clozapine sample in a quartz cell wrapped in aluminum foil for the same period of time. 2.4. Degradation kinetics study The quantitative analysis of clozapine in the tested samples was performed with the use of DAD detection at wavelength 290 nm. The developed method was validated for specificity, linearity, accuracy, precision, repeatability and sensitivity of the method. The calibration curve was obtained by plotting the peak area against the amount of the drug (STD) and studied by fitting the results to
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Table 1 Stress conditions applied to clozapine degradation. Stress conditions
Diluting solvent
Exposure conditions
Duration (h)
Acid hydrolysis Alkaline hydrolysis Neutral hydrolysis Oxidation Photolysis (UV–vis) Photolysis (UVC)
0.2 M HCl 0.2 M NaOH H2 O 1% H2 O2 H2 O H2 O
80 ◦ C 80 ◦ C 80 ◦ C Room temp. Room temp. Room temp.
0, 1, 2, 3, 4, 5, 6 0, 1, 2, 3, 4, 5, 6 0, 1, 2, 3, 4, 5, 6 0, 1, 2, 3, 4, 5, 6 0, 21, 42, 63, 84, 105, 126 0, 1, 2, 3, 4, 5, 6
linear least-squares regression. MassHunter software was used for the calculation of LOD and LOQ of the method as a signal to noise (S/N) ratio (3:1 and 10:1 respectively). The obtained calibration curve was used for the determination of degradation kinetics of clozapine in the tested conditions. In the proper time intervals (Table 1) 50 l of the tested solutions were collected and analyzed by LC-DAD/ESI-Q-TOF system. The degradation kinetics parameters: the rate constant (k) and the half-life (t1/2 ) were calculated with the use of the first-order kinetic equation: ln c = ln c0 − kt (c0 is the concentration in time 0, c is the remaining concentration). 2.5. Chemometric analysis The chemometric analysis was performed on the stressed samples (42 h for UV–vis and 6 h for the rest of the forced conditions) independently for STD and TAB. Three individual samples were prepared for each stressed condition and TOF (MS) mode was used for the registration of their chromatographic/spectral degradation profiles. The MFE (molecular feature extraction) algorithm from the Mass Hunter Qualitative Analysis software version B.06.00 (Agilent) was used for data background ion noise cleaning and to extract the list of the ions characteristic for clozapine degradation products. The MFE parameters were optimized and the following settings were applied: maximum 2 charge state of the analyzed ions, more than 5000 counts for the compound filter, isotope model: common organic molecules with peak spacing tolerance 0.0025 m/z. In order to carry out the multivariate chemometric analysis the obtained results were next exported to the MPP (Mass Profiler Professional) software version 12.6 (Agilent and Strand Life Sciences Pvt. Ltd.). With the use of this software the data was normalized and aligned and the principal component analysis (PCA) was performed in order to evaluate qualitative differences in the registered degradation profiles of clozapine. 3. Results and discussion 3.1. Quantitative analysis and degradation kinetics In order to perform a quantitative and qualitative assay in one run LC chromatographic system coupled with DAD detection and Q-TOF mass spectrometer was employed. DAD detection at wavelength 290 nm was used to the quantitative analysis and to determine the degradation kinetics of clozapine in solutions. The specificity of the method was confirmed by peak purity assessment with the use of DAD detector and the other obtained validation results confirm linearity, accuracy and precision of the proposed method (Table 2). The obtained data was used to calculate the concentration of clozapine at proper time intervals during its forced degradation in tested solutions (see Supplementary data − Table S1 in the online version at DOI: http://dx.doi.org/10.1016/j.jpba.2016. 09.007). As presented in Table 3 the decomposition of clozapine yields the first-order kinetics in all the stress conditions (r > 0.99). It was observed that clozapine is fragile towards acidic hydrolysis
Table 2 Validation results of developed method with DAD detection. Parameters
Results
Linearity Concentration range (g mL−1 ) Slope SD of slope Intercept SD of intercept r
0.4–14 5.6929 0.0684 0.9313 0.0785 0.9993
Precision − RSD (%) Intra-day (n = 12) Inter-day (n = 18)
0.63 1.79
Accuracy Recovery (%) RSD (%) LOD (g mL−1 ) LOQ (g mL−1 )
98.64 0.80 0.07 0.23
and oxidative conditions and more than 50% of parent compound was degradated. It was also noticed that clozapine is most stable under exposition to UV–vis light, however some differences were observed between STD and TAB samples. On the other hand, it was also clearly observed that in the other stress conditions no significant differences were observed between the degradation rate of bulk substance and pharmaceutical formulation. Taking this into account, it can be concluded that the formulation ingredients do not significantly affect the degradation kinetics of clozapine. 3.2. Chemometric study In order to perform the multivariate chemometric analysis all the obtained chromatographic profiles (36 chromatograms) registered in TOF (MS) mode were aligned with MPP software giving 260 entities. After a build-in MPP filtration including sample frequency, filtration by flags and setting the fold change (FC) threshold on the level not less than 4, 103 entities were finally selected for the chemometric study. The principal component analysis based on this data showed a visible categorization of all the analyzed groups of forced degradation samples (Fig. 1). Oxidative (H2 O2 ) and basic (NaOH) stressed samples stood out from the other samples while neutral (H2 O) and photo UV–vis stressed samples were very close to each other. It should be also noticed that the samples subjected to UVC irradiation visible stood out from the UV–vis stress conditions. Additionally, no differences between the pharmaceutical formulation and bulk substance samples were observed which confirm that the additional ingredients do not influence the quality composition of the obtained forced degradation profiles of clozapine. In the presented PCA analysis the first three components (PC) explained 82.06% of the total variance. 3.3. Identification of degradation products In this forced degradation study various stress conditions were used in order to compare the degradation profiles of clozapine. Similar results were obtained only in basic and neutral stress conditions
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Table 3 Degradation kinetics parameters in tested stress conditions. Stress conditions
UV–vis UVC H2 O HCl NaOH H2 O2
STD
TAB
k (h−1 )
t1/2 (h)
r
k (h−1 )
t1/2 (h)
r
0.0049 0.0357 0.0179 0.1103 0.0136 0.1266
141.5 19.4 38.6 6.2 51.1 5.4
0.9960 0.9913 0.9973 0.9977 0.9916 0.9989
0.0035 0.0405 0.0150 0.1223 0.0124 0.1464
198.0 17.1 46.2 5.6 55.9 4.7
0.9916 0.9901 0.9981 0.9977 0.9905 0.9981
Fig. 3. Q-TOF MS/MS spectrum of clozapine and corresponding fragmentation pathway.
while in the other cases some differences in the registered degradation profiles were observed (Fig. 2). On base and neutral hydrolysis the same two degradation products (D1 and D2) were found, but on acid hydrolysis D1 product was replaced by D3 degradation product. The oxidation of clozapine leads to the similar results as in the case of acidic and neutral conditions, however additional product (D6) was found. The type of radiation is an important factor in the case of photodegradation study [11] and in this study two irradiation sources − in accordance with the ICH guidelines (UV–vis) and UVC (254 nm) were compared in order to maximize the number of degradation products. It was noticed that one of the photoproduct (D5) was found only under UVC irradiation and in slight amount. As shown in Table 4 the ion masses of the above forced degradation products and the parent compound were found with very good accuracy (0.61–3.75 ppm) and the chemical formulas based on them were calculated. Based on the above data (TOF − MS spectra) as well as MS/MS fragmentation spectra the structural elucidation of the analyzed compounds was performed.
As shown in Fig. 3 MS/MS spectrum of clozapine consists of three main fragmentation ions: m/z 296.09471, 270.07959 and 84.08095 which are formed as a result of the piperazine ring breaking. Additionally, two small fragmentation ions (m/z 227.03713, 192.06761) which completed the fragmentation pathway of a parent compound of dibenzodiazepine ring were observed. Basing on this data the structure of one of the photodegradation products (D4) can be easily elucidated (Fig. 4). The measured mass (m/z 313.12164) and the generated formula (C17 H18 ClN4 ) for this compound suggest that this product is the result of the photolysis process and loss of methyl group. As shown in MS/MS spectrum of this product its fragmentation is similar to clozapine and all main characteristic ions for this compound are observed. The main difference in this case is the presence of m/z 70.06554 fragmentation ion which confirms that N-dealkylation process took place on the piperazine ring. The degradation product D1 is characterized by 16 Da mass higher (m/z 343.13116) than clozapine which suggests that this product has an extra oxygen atom. In these conditions first the possibility of the formation of N-oxide derivatives was consid-
276
Table 4 Q-TOF accurate mass elemental composition and MS/MS fragmentation of the analyzed compounds. Observed in stress condition
CLO
Retention time (min)
Measured mass (m/z)
Theoretical mass (m/z)
Mass error (ppm)
Molecular formula [M + H]+
MS/MS fragmentation (m/z)
Fragmentation ion formula [M + H]+
5.20
327.13741
327.13710
0.95
C18 H20 ClN4
296.09471 270.07959 227.03713 192.06761 84.08095 325.12073 299.10489 256.0637 243.05546 99.09235 85.0775 86.09658 59.05000 327.13629 245.04764 101.10738 296.29583 270.07948 244.0618 227.03869 192.06841 70.06554 250.09671 209.06951 97.08121 71.08561 57.06993 100.09954 84.95994
C17 H15 ClN3 C15 H13 ClN3 C13 H8 ClN2 C13 H8 N2 C5 H10 N C18 H18 ClN4 C16 H16 ClN4 C14 H11 ClN3 C13 H10 ClN3 C5 H11 N2 C4 H9 N2 C5 H12 N C3 H9 N C18 H20 ClN4 C13 H10 ClN2 O C5 H13 N2 C17 H15 ClN3 C15 H13 ClN3 C13 H11 ClN3 C13 H8 ClN2 C13 H8 N2 C4 H8 N C15 H12 N3 O C13 H9 N2 O C5 H9 N2 C4 H9 N C3 H7 N C5 H12 N2 C5 H11 N
D1
UV–vis, UVC, H2 O, H2 O2 , NaOH
5.80
343.13116
343.13202
2.50
C18 H20 ClN4 O
D2
0.52
101.10770
101.10732
3.75
C5 H13 N2
D3
UV–vis, UVC, H2 O, H2 O2 , NaOH, HCl HCl
4.90
345.14804
345.14767
1.07
C18 H22 ClN4 O
D4
UV–vis, UVC
4.75
313.12164
313.12145
0.61
C17 H18 ClN4
D5
UVC
2.90
307.15555
307.15534
0.68
C18 H19 N4 O
D6
H2 O2
0.56
117.10208
117.10224
1.37
C5 H13 N2 O
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Fig. 4. Q-TOF MS/MS spectrum and fragmentation pathway of degradation product D4.
ered. As shown in Fig. 5 the obtained fragmentation spectrum for this product differs significantly from MS/MS spectrum of the parent compound, however the registered fragmentation ions indicate that the N-oxide bond was formed with nitrogen in the piperazine ring and not with the dibenzodiazepine ring. It should be noticed that D1 product was found in the all tested conditions except for acid hydrolysis and it is the main degradation product of oxidative stress. The measured mass of the second forced degradation product D2 (m/z 101.10770), which was observed in all stress conditions, enabled the generation of C5 H13 N2 formula which corresponds with methyl-piperazinyl moiety of clozapine. The obtained MS/MS fragmentation spectrum of this product (Fig. 6) exactly confirmed the proposed structure. Although this product was identified in all the tested conditions it should be noticed that at the same time no dibenzodiazepine ring based products were found in the stressed samples. This suggests that when the forced hydrolysis of the parent compound is observed it leads to the destruction of the main tricyclic ringand only piperazine derivatives can be found. The degradation product D3 was found as m/z 345.14767 ion which is higher than clozapine accurate mass by 18 Da. As shown in Fig. 7 this product was elucidated as a hydroxylated derivative of the parent compound and the registered main fragmentation ion m/z 245.04764 confirmed that hydroxylation had taken place presumably at nitrogen in position 10 of the dibenzodiazepine ring. This product is characteristic only for acid hydrolysis in aqueous
conditions which leads to double bond C N cleavage and its substitution by a hydroxyl group. Although in these conditions the hydrolysis of a 7-membered ring and the formation of an opened amide ring would be primarily expected, the presence of ion m/z 245.04764 in all the registered MS/MS spectra (various CID energy were tested) and at the same time the absence of fragmentation ions characteristic for the dibenzodiazepine ring dissociation deflate this hipothesis. In Fig. 8 MS/MS spectrum and the fragmentation pathway of D5 product, which was observed only under irradiation of UVC light, are presented. Basing on the isotopic envelope of the parent ion and its exact protonated mass (m/z 307.15555) first of all the loss of chlorine atom was noticed and also additional oxygen atom was observed. The fragmentation spectrum shows that chlorine atom was replaced by oxygen with a double bond rearrangement in dibenzodiazepine ring which confirms the presence of ions: m/z 250.09671 and 209.06951. This kind of reactions is well known in the case of forced photodegradation process with the use of high-pressure mercury lamp [12]. The measured mass of the D6 product (m/z 117.10224) enabled the generation of C5 H13 N2 O formula which corresponds to methylpiperazinyl moiety with extra oxygen atom. This compound can be formed as a secondary degradation product from D2 product in the presence of hydrogen peroxide as a strong oxidation agent. The obtained MS/MS fragmentation spectrum of this product (Fig. 9) confirmed the proposed structure. The presence of ion
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Fig. 5. Q-TOF MS/MS spectrum and fragmentation pathway of degradation product D1.
Fig. 6. Q-TOF MS/MS spectrum and fragmentation pathway of degradation product D2.
m/z 100.09954 in this spectrum legitimize that hydroxylation had taken place at nitrogen in position 1 of the piperazine ring. The proposed forced degradation pathway of clozapine in tested stress conditions is presented in Fig. 10. 4. Conclusion The degradation behavior of clozapine under hydrolytic (acid, base and neutral), oxidative and photolytic (as per ICH guidelines and UVC) stress was studied. The decomposition of the drug yields the first-order kinetic reaction in all stress conditions and
it is fragile towards acidic hydrolysis and oxidative conditions. Six degradation products were found and basing on MS/MS fragmentation spectra their structural elucidation was performed. Two main degradation products were formed as an effect of N-oxidation (D1) and direct hydrolysis (D2) of the parent compound. The use of forced UVC photodegradation allowed to indentify one additional degradation product (D5) which was not observed under typical photolysis conditions. The multivariate chemometric analysis (PCA) allowed a preliminary characterization of the registered degradation profiles and led to conclusion that the pharmaceutical formulation ingredients do
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Fig. 7. Q-TOF MS/MS spectrum and fragmentation pathway of degradation product D3.
Fig. 8. Q-TOF MS/MS spectrum and fragmentation pathway of degradation product D5.
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Fig. 9. Q-TOF MS/MS spectrum and fragmentation pathway of degradation product D6.
Fig. 10. Forced degradation pathway of clozapine in tested conditions.
not affect the quality composition of the obtained forced degradation profiles of clozapine. Acknowledgements The paper was developed with the use of the equipment purchased within the Project “The equipment of innovative laboratories doing research on new medicines used in the therapy of civilization and neoplastic diseases” within the Operational Program Development of Eastern Poland 2007–2013, Priority Axis I Modern Economy, Operations I.3 Innovation Promotion. References [1] J. Crilly, The history of clozapine and its emergence in the US market: a review and analysis, Hist. Psychiatry 18 (2007) 39–60. [2] H.Y. Meltzer, An overview of the mechanism of action of clozapine, J. Clin. Psychiatry 55 (1994) 47–52. [3] D. Miller, Review and management of clozapine side effects, J. Clin. Psychiatry 61 (2000) 14–17. [4] S. Warnez, S. Alessi-Severini, Clozapine: a review of clinical practice guidelines and prescribing trends, Psychiatry 14 (2014) 1–5.
[5] ICH guideline, Q1A (R2) stability yesting of new drug substances and products, in: International Conference on Harmonisation, IFPMA, Geneva, Switzerland, 2003. [6] ICH guideline, Q1 B photostability testing of new drug substances and products, in: International Conference on Harmonisation, IFPMA, Geneva, Switzerland, 1996. [7] D. Jacobson-Kram, T. McGovern, Toxicological overview of impurities in pharmaceutical products, Adv. Drug Deliv. Rev. 59 (2007) 38–42. [8] Z. Zaheer, M. Farooqui, S.R. Dhaneshwar, Stability-indicating high performance thin layer chromatographic determination of clozapine in tablet dosage form, J. Pharm. Sci. Res. 1 (2009) 158–166. [9] N.Y. Hasan, M.A. Elkawy, B.E. Elzeany, N.E. Wagieh, Stability indicating methods for the determination of clozapine, J. Pharm. Biomed. Anal. 30 (2002) 35–47. [10] S.E. Walker, D. Baker, S. Law, Stability of clozapine stored in oral suspension vehicles at room temperature, Can. J. Hosp. Pharm. 58 (2005) 279–284. ´ [11] R. Skibinski, Ł. Komsta, T. Inglot, Characterization of paliperidone photodegradation products by LC-Q-TOF multistage mass spectrometry, Biomed. Chromatogr. 30 (2016) 894–901. [12] R. Andersin, J. Ovaskainen, S. Kaltia, Photochemical decomposition of midazolam. III — Isolation and identification of products in aqueous solutions, J. Pharm. Biomed. Anal. 12 (1994) 165–172.
Contents lists available at ScienceDirect
Journal of Pharmaceutical and Biomedical Analysis journal homepage: www.elsevier.com/locate/jpba
Characterization of forced degradation products of clozapine by LC-DAD/ESI-Q-TOF ∗ ´ ´ Robert Skibinski , Jakub Trawinski, Łukasz Komsta, Katarzyna Bajda Department of Medicinal Chemistry, Medical University of Lublin, Jaczewskiego 4, 20-090 Lublin, Poland
a r t i c l e
i n f o
Article history: Received 28 June 2016 Received in revised form 29 August 2016 Accepted 3 September 2016 Available online 4 September 2016 Keywords: Mass spectrometry UHPLC Stressed degradation Photodegradation Clozapine
a b s t r a c t Forced degradation of clozapine in solutions under acidic, basic, neutral, photo UV–vis, photo UVC and oxidative stress conditions was investigated and structural elucidation of its degradation products was performed with the use of the UHPLC-DAD system coupled with accurate hybrid ESI-Q-TOF mass spectrometer. The developed method allows to collect all essential data for the determination of degradation kinetics and for the structural elucidation of the formed products. Six degradation products were found and their masses and formulas were obtained with high accuracy (0.61–3.75 ppm). For all the analyzed compounds MS/MS fragmentation spectra were also obtained allowing structural elucidation of the unknown degradation products. It was found that the decomposition of clozapine yields the first-order kinetic reaction in all stress conditions and it is fragile towards acidic hydrolysis and oxidative conditions. Additionally, PCA analysis of registered TOF (MS) forced degradation profiles of clozapine shows no differences between the samples obtained from pharmaceutical formulation and bulk substance. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Clozapine (8-chloro-11-(4-methylpiperazin-1-yl)-5Hdibenzo[b,e][1,4]diazepine) is the first atypical antipsychotic medication introduced to the market in 1960s as an effective agent to schizophrenia treatment [1]. Its pharmacological activity is based on the selective 5-HT2A and D4 receptor antagonist together with its weak D2 receptor blocking [2]. Clozapine is characterized by superior efficacy in relieving the positive and negative symptoms in treatment-resistant schizophrenic patients with minimal extrapyramidal side effects, however with the risk of agranulocytosis [3,4]. Stress testing studies are an integral part of the stability testing of drugs and must be considered during the development and registration process of pharmaceuticals. The degradation of drugs is an important part of investigation because this process can result in the loss of drug effectiveness as well as it can lead to additional adverse effects due to the formation of toxic degradation products. From this point of view it is very important to know what products (impurities) are formed from the medicines during the degradation process and what their exact chemical structure is. This information can be very useful for the manufacturing, quality control, storage and administration of pharmaceuticals [5–7].
∗ Corresponding author. ´ E-mail address: [email protected] (R. Skibinski). http://dx.doi.org/10.1016/j.jpba.2016.09.007 0731-7085/© 2016 Elsevier B.V. All rights reserved.
The degradation and stability study of clozapine was not studied exactly so far and only some papers concerning the development of stability-indicating method for the analysis of this drug with the use of TLC [8,9], LC-UV and UV–vis spectrophotometry [9] are published. In these papers no structural elucidation of degradation products was performed and only Hasan et al. [9] noticed that under acidic hydrolysis one degradation product − N-methylpiperazine was obtained. The stability of clozapine was also investigated in oral suspension at room temperature by the use of LC-UV method [10]. Hence, it is necessary to carry out the forced degradation study of clozapine including the structure elucidation of the formed products. For this purpose a new analytical method with the use of UHPLC-DAD system coupled with accurate hybrid ESI-Q-TOF mass spectrometer was developed. Additionally, the multivariate chemometric analysis (PCA) of forced degradation profiles of clozapine from bulk substance and pharmaceutical formulation was performed.
2. Experimental 2.1. Chemicals and reagents Clozapine and water for LC–MS Ultra grade were obtained from Sigma-Aldrich (St. Louis, USA). Methanol hypergrade for LC–MS and acetonitrile gradient grade were purchased from Merck (Darmstadt, Germany) and 98% formic acid for mass spectroscopy was
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obtained from Fluka (Taufkirchen, Germany). All analytical grade reagents (hydrochloric acid, sodium hydroxide, 30% hydrogen peroxide) were purchased from POCh (Gliwice, Poland). Klozapol tablets (100 mg of clozapine) were obtained from Anpharm (Warsaw, Poland). 2.2. LC-DAD/ESI-Q-TOF analysis LC–MS analysis was performed with the use of Agilent AccurateMass Q-TOF LC/MS G6520 B system with dual electrospray (DESI) ionization source and Infinity 1290 ultra-high-pressure liquid chromatography system consisting of: binary pomp G4220A, FC/ALS thermostat G1330B, autosampler G4226A, DAD detector G4212A, TCC G1316C module and Zorbax Eclipse-C18 (2.1 × 50 mm, dp = 1.8 m) HD column (Agilent Technologies, Santa Clara, USA). A mixture of methanol (A) and water (B) with addition of 0.1% solution of formic acid in both media was used as a mobile phase. The gradient elution was carried out at constant flow 0.3 ml/min from 20%A (80%B) 0 − 1 min and then 20%A to 70%A 1–7.5 min 1 min post time was performed to return to initial conditions. The injection volume was 1 l and the column temperature was maintained at 35 ◦ C. MassHunter workstation software in version B.04.00 was used for the control of the system, data acquisition, qualitative and quantitative analysis. The optimization of the instrument conditions was set out from the proper tuning of Q-TOF detector in a positive mode with the use of Agilent ESI-L tuning mix in the extended dynamic range (2 GHz). The following instrument settings were applied: gas temp.: 300 ◦ C, drying gas: 10 l/min, nebulizer pressure: 40 psig, capillary voltage: 3000 V, fragmentor voltage: 110 V, skimmer voltage: 65 V, octopole 1 RF voltage: 250 V. Data acquisition was performed in centroids with the use of TOF (MS) and also targeted MS/MS mode. The spectral parameters for both modes were: mass range: 50–950 m/z and the acquisition rate: 1.2 spectra/s. The collision energy (CID) for MS/MS experiments was ranged: 7.8–25.7 eV. To ensure accuracy in masses measurements, a reference mass correction was used and masses 121.050873 and 922.009798 were used as lock masses. DAD detector (200–400 nm) was also used for the additional monitoring and quantitation analysis of clozapine ( = 290 nm).
Fig. 1. The 3D PCA plot of forced degradation profiles of standard (squares) and pharmaceutical formulation (triangles) samples of clozapine.
2.3. Forced degradation studies Forced degradation studies were performed independently for the bulk substance (STD) and pharmaceutical formulation (TAB) of clozapine. Stock STD and TAB solutions of clozapine were prepared in acetonitrile at concentration 500 g mL−1 . In the case of TAB solution the equivalent of 12,5 mg of clozapine from mortared 20 Klozapol tablets was transferred to 25 ml volumetric flask and after the addition of acetonitrile extracted by shaker. The obtained suspension was centrifuged and next used as a stock solution. The working solutions were prepared by diluting stock solutions using the proper solvent to obtain a final concentration of 10 g mL−1 and next stressed under hydrolytic, oxidative and photolytic conditions (Table 1). All the hydrolytic and oxidative tests were performed using 10 ml of working solution placed in hermetically sealed glass vials. For the photodegradation tests the working solutions were placed in a quartz caped cells (l = 1 cm) mounted horizontally and irradiated with UV–vis or UVC radiation. The distance between the lamp and the samples was 10 cm in both cases. As a UV–vis source a photostability chamber Atlas Suntest CPS+ (Linsengericht, Germany) with full UV–vis spectrum (ID65) according to ICH guidelines was used. The irradiance was set to 250 W/m2 which corresponds to the dose of 900 kJ/m2 /h. Under these conditions the recommended ICH dose (1200000 lx of light intensity) was
Fig. 2. TOF total ion chromatograms obtained under acidic (A), basic (B), neutral (C), photo UV–vis (D), photo UVC (E) and oxidative (F) stress conditions.
reached after 21 h of irradiation. As a UVC source Haland HA-05 (Warsaw, Poland) ultraviolet laboratory lamp equipped with 6W quartz ultraviolet tube emitting mercury spectrum with 254 nm principal line was used. The average UVC irradiation intensity was 7.5 W/m2 . The dark control samples were also performed for both photostability experiments by exposing the clozapine sample in a quartz cell wrapped in aluminum foil for the same period of time. 2.4. Degradation kinetics study The quantitative analysis of clozapine in the tested samples was performed with the use of DAD detection at wavelength 290 nm. The developed method was validated for specificity, linearity, accuracy, precision, repeatability and sensitivity of the method. The calibration curve was obtained by plotting the peak area against the amount of the drug (STD) and studied by fitting the results to
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Table 1 Stress conditions applied to clozapine degradation. Stress conditions
Diluting solvent
Exposure conditions
Duration (h)
Acid hydrolysis Alkaline hydrolysis Neutral hydrolysis Oxidation Photolysis (UV–vis) Photolysis (UVC)
0.2 M HCl 0.2 M NaOH H2 O 1% H2 O2 H2 O H2 O
80 ◦ C 80 ◦ C 80 ◦ C Room temp. Room temp. Room temp.
0, 1, 2, 3, 4, 5, 6 0, 1, 2, 3, 4, 5, 6 0, 1, 2, 3, 4, 5, 6 0, 1, 2, 3, 4, 5, 6 0, 21, 42, 63, 84, 105, 126 0, 1, 2, 3, 4, 5, 6
linear least-squares regression. MassHunter software was used for the calculation of LOD and LOQ of the method as a signal to noise (S/N) ratio (3:1 and 10:1 respectively). The obtained calibration curve was used for the determination of degradation kinetics of clozapine in the tested conditions. In the proper time intervals (Table 1) 50 l of the tested solutions were collected and analyzed by LC-DAD/ESI-Q-TOF system. The degradation kinetics parameters: the rate constant (k) and the half-life (t1/2 ) were calculated with the use of the first-order kinetic equation: ln c = ln c0 − kt (c0 is the concentration in time 0, c is the remaining concentration). 2.5. Chemometric analysis The chemometric analysis was performed on the stressed samples (42 h for UV–vis and 6 h for the rest of the forced conditions) independently for STD and TAB. Three individual samples were prepared for each stressed condition and TOF (MS) mode was used for the registration of their chromatographic/spectral degradation profiles. The MFE (molecular feature extraction) algorithm from the Mass Hunter Qualitative Analysis software version B.06.00 (Agilent) was used for data background ion noise cleaning and to extract the list of the ions characteristic for clozapine degradation products. The MFE parameters were optimized and the following settings were applied: maximum 2 charge state of the analyzed ions, more than 5000 counts for the compound filter, isotope model: common organic molecules with peak spacing tolerance 0.0025 m/z. In order to carry out the multivariate chemometric analysis the obtained results were next exported to the MPP (Mass Profiler Professional) software version 12.6 (Agilent and Strand Life Sciences Pvt. Ltd.). With the use of this software the data was normalized and aligned and the principal component analysis (PCA) was performed in order to evaluate qualitative differences in the registered degradation profiles of clozapine. 3. Results and discussion 3.1. Quantitative analysis and degradation kinetics In order to perform a quantitative and qualitative assay in one run LC chromatographic system coupled with DAD detection and Q-TOF mass spectrometer was employed. DAD detection at wavelength 290 nm was used to the quantitative analysis and to determine the degradation kinetics of clozapine in solutions. The specificity of the method was confirmed by peak purity assessment with the use of DAD detector and the other obtained validation results confirm linearity, accuracy and precision of the proposed method (Table 2). The obtained data was used to calculate the concentration of clozapine at proper time intervals during its forced degradation in tested solutions (see Supplementary data − Table S1 in the online version at DOI: http://dx.doi.org/10.1016/j.jpba.2016. 09.007). As presented in Table 3 the decomposition of clozapine yields the first-order kinetics in all the stress conditions (r > 0.99). It was observed that clozapine is fragile towards acidic hydrolysis
Table 2 Validation results of developed method with DAD detection. Parameters
Results
Linearity Concentration range (g mL−1 ) Slope SD of slope Intercept SD of intercept r
0.4–14 5.6929 0.0684 0.9313 0.0785 0.9993
Precision − RSD (%) Intra-day (n = 12) Inter-day (n = 18)
0.63 1.79
Accuracy Recovery (%) RSD (%) LOD (g mL−1 ) LOQ (g mL−1 )
98.64 0.80 0.07 0.23
and oxidative conditions and more than 50% of parent compound was degradated. It was also noticed that clozapine is most stable under exposition to UV–vis light, however some differences were observed between STD and TAB samples. On the other hand, it was also clearly observed that in the other stress conditions no significant differences were observed between the degradation rate of bulk substance and pharmaceutical formulation. Taking this into account, it can be concluded that the formulation ingredients do not significantly affect the degradation kinetics of clozapine. 3.2. Chemometric study In order to perform the multivariate chemometric analysis all the obtained chromatographic profiles (36 chromatograms) registered in TOF (MS) mode were aligned with MPP software giving 260 entities. After a build-in MPP filtration including sample frequency, filtration by flags and setting the fold change (FC) threshold on the level not less than 4, 103 entities were finally selected for the chemometric study. The principal component analysis based on this data showed a visible categorization of all the analyzed groups of forced degradation samples (Fig. 1). Oxidative (H2 O2 ) and basic (NaOH) stressed samples stood out from the other samples while neutral (H2 O) and photo UV–vis stressed samples were very close to each other. It should be also noticed that the samples subjected to UVC irradiation visible stood out from the UV–vis stress conditions. Additionally, no differences between the pharmaceutical formulation and bulk substance samples were observed which confirm that the additional ingredients do not influence the quality composition of the obtained forced degradation profiles of clozapine. In the presented PCA analysis the first three components (PC) explained 82.06% of the total variance. 3.3. Identification of degradation products In this forced degradation study various stress conditions were used in order to compare the degradation profiles of clozapine. Similar results were obtained only in basic and neutral stress conditions
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Table 3 Degradation kinetics parameters in tested stress conditions. Stress conditions
UV–vis UVC H2 O HCl NaOH H2 O2
STD
TAB
k (h−1 )
t1/2 (h)
r
k (h−1 )
t1/2 (h)
r
0.0049 0.0357 0.0179 0.1103 0.0136 0.1266
141.5 19.4 38.6 6.2 51.1 5.4
0.9960 0.9913 0.9973 0.9977 0.9916 0.9989
0.0035 0.0405 0.0150 0.1223 0.0124 0.1464
198.0 17.1 46.2 5.6 55.9 4.7
0.9916 0.9901 0.9981 0.9977 0.9905 0.9981
Fig. 3. Q-TOF MS/MS spectrum of clozapine and corresponding fragmentation pathway.
while in the other cases some differences in the registered degradation profiles were observed (Fig. 2). On base and neutral hydrolysis the same two degradation products (D1 and D2) were found, but on acid hydrolysis D1 product was replaced by D3 degradation product. The oxidation of clozapine leads to the similar results as in the case of acidic and neutral conditions, however additional product (D6) was found. The type of radiation is an important factor in the case of photodegradation study [11] and in this study two irradiation sources − in accordance with the ICH guidelines (UV–vis) and UVC (254 nm) were compared in order to maximize the number of degradation products. It was noticed that one of the photoproduct (D5) was found only under UVC irradiation and in slight amount. As shown in Table 4 the ion masses of the above forced degradation products and the parent compound were found with very good accuracy (0.61–3.75 ppm) and the chemical formulas based on them were calculated. Based on the above data (TOF − MS spectra) as well as MS/MS fragmentation spectra the structural elucidation of the analyzed compounds was performed.
As shown in Fig. 3 MS/MS spectrum of clozapine consists of three main fragmentation ions: m/z 296.09471, 270.07959 and 84.08095 which are formed as a result of the piperazine ring breaking. Additionally, two small fragmentation ions (m/z 227.03713, 192.06761) which completed the fragmentation pathway of a parent compound of dibenzodiazepine ring were observed. Basing on this data the structure of one of the photodegradation products (D4) can be easily elucidated (Fig. 4). The measured mass (m/z 313.12164) and the generated formula (C17 H18 ClN4 ) for this compound suggest that this product is the result of the photolysis process and loss of methyl group. As shown in MS/MS spectrum of this product its fragmentation is similar to clozapine and all main characteristic ions for this compound are observed. The main difference in this case is the presence of m/z 70.06554 fragmentation ion which confirms that N-dealkylation process took place on the piperazine ring. The degradation product D1 is characterized by 16 Da mass higher (m/z 343.13116) than clozapine which suggests that this product has an extra oxygen atom. In these conditions first the possibility of the formation of N-oxide derivatives was consid-
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Table 4 Q-TOF accurate mass elemental composition and MS/MS fragmentation of the analyzed compounds. Observed in stress condition
CLO
Retention time (min)
Measured mass (m/z)
Theoretical mass (m/z)
Mass error (ppm)
Molecular formula [M + H]+
MS/MS fragmentation (m/z)
Fragmentation ion formula [M + H]+
5.20
327.13741
327.13710
0.95
C18 H20 ClN4
296.09471 270.07959 227.03713 192.06761 84.08095 325.12073 299.10489 256.0637 243.05546 99.09235 85.0775 86.09658 59.05000 327.13629 245.04764 101.10738 296.29583 270.07948 244.0618 227.03869 192.06841 70.06554 250.09671 209.06951 97.08121 71.08561 57.06993 100.09954 84.95994
C17 H15 ClN3 C15 H13 ClN3 C13 H8 ClN2 C13 H8 N2 C5 H10 N C18 H18 ClN4 C16 H16 ClN4 C14 H11 ClN3 C13 H10 ClN3 C5 H11 N2 C4 H9 N2 C5 H12 N C3 H9 N C18 H20 ClN4 C13 H10 ClN2 O C5 H13 N2 C17 H15 ClN3 C15 H13 ClN3 C13 H11 ClN3 C13 H8 ClN2 C13 H8 N2 C4 H8 N C15 H12 N3 O C13 H9 N2 O C5 H9 N2 C4 H9 N C3 H7 N C5 H12 N2 C5 H11 N
D1
UV–vis, UVC, H2 O, H2 O2 , NaOH
5.80
343.13116
343.13202
2.50
C18 H20 ClN4 O
D2
0.52
101.10770
101.10732
3.75
C5 H13 N2
D3
UV–vis, UVC, H2 O, H2 O2 , NaOH, HCl HCl
4.90
345.14804
345.14767
1.07
C18 H22 ClN4 O
D4
UV–vis, UVC
4.75
313.12164
313.12145
0.61
C17 H18 ClN4
D5
UVC
2.90
307.15555
307.15534
0.68
C18 H19 N4 O
D6
H2 O2
0.56
117.10208
117.10224
1.37
C5 H13 N2 O
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Fig. 4. Q-TOF MS/MS spectrum and fragmentation pathway of degradation product D4.
ered. As shown in Fig. 5 the obtained fragmentation spectrum for this product differs significantly from MS/MS spectrum of the parent compound, however the registered fragmentation ions indicate that the N-oxide bond was formed with nitrogen in the piperazine ring and not with the dibenzodiazepine ring. It should be noticed that D1 product was found in the all tested conditions except for acid hydrolysis and it is the main degradation product of oxidative stress. The measured mass of the second forced degradation product D2 (m/z 101.10770), which was observed in all stress conditions, enabled the generation of C5 H13 N2 formula which corresponds with methyl-piperazinyl moiety of clozapine. The obtained MS/MS fragmentation spectrum of this product (Fig. 6) exactly confirmed the proposed structure. Although this product was identified in all the tested conditions it should be noticed that at the same time no dibenzodiazepine ring based products were found in the stressed samples. This suggests that when the forced hydrolysis of the parent compound is observed it leads to the destruction of the main tricyclic ringand only piperazine derivatives can be found. The degradation product D3 was found as m/z 345.14767 ion which is higher than clozapine accurate mass by 18 Da. As shown in Fig. 7 this product was elucidated as a hydroxylated derivative of the parent compound and the registered main fragmentation ion m/z 245.04764 confirmed that hydroxylation had taken place presumably at nitrogen in position 10 of the dibenzodiazepine ring. This product is characteristic only for acid hydrolysis in aqueous
conditions which leads to double bond C N cleavage and its substitution by a hydroxyl group. Although in these conditions the hydrolysis of a 7-membered ring and the formation of an opened amide ring would be primarily expected, the presence of ion m/z 245.04764 in all the registered MS/MS spectra (various CID energy were tested) and at the same time the absence of fragmentation ions characteristic for the dibenzodiazepine ring dissociation deflate this hipothesis. In Fig. 8 MS/MS spectrum and the fragmentation pathway of D5 product, which was observed only under irradiation of UVC light, are presented. Basing on the isotopic envelope of the parent ion and its exact protonated mass (m/z 307.15555) first of all the loss of chlorine atom was noticed and also additional oxygen atom was observed. The fragmentation spectrum shows that chlorine atom was replaced by oxygen with a double bond rearrangement in dibenzodiazepine ring which confirms the presence of ions: m/z 250.09671 and 209.06951. This kind of reactions is well known in the case of forced photodegradation process with the use of high-pressure mercury lamp [12]. The measured mass of the D6 product (m/z 117.10224) enabled the generation of C5 H13 N2 O formula which corresponds to methylpiperazinyl moiety with extra oxygen atom. This compound can be formed as a secondary degradation product from D2 product in the presence of hydrogen peroxide as a strong oxidation agent. The obtained MS/MS fragmentation spectrum of this product (Fig. 9) confirmed the proposed structure. The presence of ion
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Fig. 5. Q-TOF MS/MS spectrum and fragmentation pathway of degradation product D1.
Fig. 6. Q-TOF MS/MS spectrum and fragmentation pathway of degradation product D2.
m/z 100.09954 in this spectrum legitimize that hydroxylation had taken place at nitrogen in position 1 of the piperazine ring. The proposed forced degradation pathway of clozapine in tested stress conditions is presented in Fig. 10. 4. Conclusion The degradation behavior of clozapine under hydrolytic (acid, base and neutral), oxidative and photolytic (as per ICH guidelines and UVC) stress was studied. The decomposition of the drug yields the first-order kinetic reaction in all stress conditions and
it is fragile towards acidic hydrolysis and oxidative conditions. Six degradation products were found and basing on MS/MS fragmentation spectra their structural elucidation was performed. Two main degradation products were formed as an effect of N-oxidation (D1) and direct hydrolysis (D2) of the parent compound. The use of forced UVC photodegradation allowed to indentify one additional degradation product (D5) which was not observed under typical photolysis conditions. The multivariate chemometric analysis (PCA) allowed a preliminary characterization of the registered degradation profiles and led to conclusion that the pharmaceutical formulation ingredients do
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Fig. 7. Q-TOF MS/MS spectrum and fragmentation pathway of degradation product D3.
Fig. 8. Q-TOF MS/MS spectrum and fragmentation pathway of degradation product D5.
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Fig. 9. Q-TOF MS/MS spectrum and fragmentation pathway of degradation product D6.
Fig. 10. Forced degradation pathway of clozapine in tested conditions.
not affect the quality composition of the obtained forced degradation profiles of clozapine. Acknowledgements The paper was developed with the use of the equipment purchased within the Project “The equipment of innovative laboratories doing research on new medicines used in the therapy of civilization and neoplastic diseases” within the Operational Program Development of Eastern Poland 2007–2013, Priority Axis I Modern Economy, Operations I.3 Innovation Promotion. References [1] J. Crilly, The history of clozapine and its emergence in the US market: a review and analysis, Hist. Psychiatry 18 (2007) 39–60. [2] H.Y. Meltzer, An overview of the mechanism of action of clozapine, J. Clin. Psychiatry 55 (1994) 47–52. [3] D. Miller, Review and management of clozapine side effects, J. Clin. Psychiatry 61 (2000) 14–17. [4] S. Warnez, S. Alessi-Severini, Clozapine: a review of clinical practice guidelines and prescribing trends, Psychiatry 14 (2014) 1–5.
[5] ICH guideline, Q1A (R2) stability yesting of new drug substances and products, in: International Conference on Harmonisation, IFPMA, Geneva, Switzerland, 2003. [6] ICH guideline, Q1 B photostability testing of new drug substances and products, in: International Conference on Harmonisation, IFPMA, Geneva, Switzerland, 1996. [7] D. Jacobson-Kram, T. McGovern, Toxicological overview of impurities in pharmaceutical products, Adv. Drug Deliv. Rev. 59 (2007) 38–42. [8] Z. Zaheer, M. Farooqui, S.R. Dhaneshwar, Stability-indicating high performance thin layer chromatographic determination of clozapine in tablet dosage form, J. Pharm. Sci. Res. 1 (2009) 158–166. [9] N.Y. Hasan, M.A. Elkawy, B.E. Elzeany, N.E. Wagieh, Stability indicating methods for the determination of clozapine, J. Pharm. Biomed. Anal. 30 (2002) 35–47. [10] S.E. Walker, D. Baker, S. Law, Stability of clozapine stored in oral suspension vehicles at room temperature, Can. J. Hosp. Pharm. 58 (2005) 279–284. ´ [11] R. Skibinski, Ł. Komsta, T. Inglot, Characterization of paliperidone photodegradation products by LC-Q-TOF multistage mass spectrometry, Biomed. Chromatogr. 30 (2016) 894–901. [12] R. Andersin, J. Ovaskainen, S. Kaltia, Photochemical decomposition of midazolam. III — Isolation and identification of products in aqueous solutions, J. Pharm. Biomed. Anal. 12 (1994) 165–172.