Characterization of forced degradation products of toloxatone by LC-ESI-MS/MS

Abstract

Forced degradation of toloxatone in solutions under basic, acidic, 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 UHPLC system coupled ESI-Q-TOF mass spectrometer. Eight degradation products were found and their masses and formulas were obtained with high accuracy (0.09–3.79 ppm). The structure of unknown degradation products were elucidated from MS/MS fragmentation spectra of all analyzed compounds. Additionally, whole signals of decomposed substances were compared chemometrically. It was found that toloxatone is fragile towards basic hydrolysis, oxidative conditions and UVC irradiation. Finally, the toxicity of transformation products was computationally evaluated and compared in multivariate manner.

1. Introduction

Toloxatone (5-(hydroxymethyl)-3-(3-methylphenyl)-1,3-oxazolidin-2-one) is the third generation of monoamine oxidase inhibitors (MAO) introduced to the market in the late 1980s as an effective agent to major depression. Its pharmacological activity is based on the selective and reversible inhibition of monoamine oxidase type A (RIMA) and is characterized by minimal adverse side effects in comparison to previous two generations of MAO inhibitors (Moureaul et al., 1992, Moureau et al., 1995). It was also reported that the antidepressant efficiency of toloxatone is similar to the most popular RIMA – moclobemide, however, its onset of action is slower (Bonnet, 2003).

In the analytical aspect toloxatone was only studied in the biological materials for the qualification as well as identification of this drug. HPLC with UV detection was the most often used method for the determination of toloxatone in human plasma (Duverneuil et al., 2003, Provost et al., 1992), rabbit plasma and cerebrospinal fluid (Kaltenbach et al., 1999). HPLC with MS/MS detection was used for the qualification of the drug in whole blood (Titier et al., 2007) and GLC method was also used for its determination in plasma (Vajta et al., 1983). The other chromatographic method – TLC was also applied for the identification of toloxatone and its metabolites in urine (Vajta et al., 1984).

It should be noticed that degradation study of drugs is an important part of stability testing of medicines, as decomposed drugs can lose their effectiveness as well as they can gain additional adverse effects. Therefore it is very important to know what transformation products are formed during the degradation process. This data can be very useful for the manufacturing, quality control, storage and administration of pharmaceuticals (ICH guideline, 1996, ICH guideline, 2003, Jacobson-Kram and McGovern, 2007).

Hence, it is necessary to perform the forced degradation study of toloxatone including the structure elucidation of the formed products. For this purpose a new analytical method using UHPLC system coupled with accurate hybrid ESI-MS/MS spectrometer was developed. Additionally, the multivariate chemometric analyses (PCA) of the forced degradation profiles of toloxatone as well as in silico toxicity of the identified transformation products were performed.

2. Experimental

2.1. Chemicals and reagents

The following chemicals were used: toloxatone (Sigma Aldrich, St Louis, USA), LC-MS grade water (Sigma Aldrich, St Louis, USA) and 30% hydrogen peroxide of trace analysis grade (Sigma Aldrich, St Louis, USA), acetonitrile hypergrade for LC-MS (Merck, Darmstadt, Germany, 98% formic acid of mass spectroscopy grade (Fluka, Taufkirchen, Germany). All other analytical grade reagents (hydrochloric acid, sodium hydroxide) were purchased from POCh (Gliwice, Poland).

2.2. LC-ESI-MS/MS analysis

The LC-MS/MS analysis was performed on Agilent Accurate-Mass Q-TOF LC/MS G6520B system with dual electrospray (DESI) ionization source and Infinity 1290 ultra-high-pressure liquid chromatography system consisting of: binary pump G4220A, FC/ALS thermostat G1330B, autosampler G4226A, DAD detector G4212A, TCC G1316C module and Zorbax Eclipse-C18 (2.1 × 50 mm, dp = 1.8 μm) HD column (Agilent Technologies, Santa Clara, USA). A mixture of acetonitrile (A) and water (B) with addition of 0.1% solution of formic acid in both media was used as a mobile phase. The isocratic elution was carried out at constant flow 0.5 ml/min at 10%A and 90%B. The injection volume was 5 µl 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 started 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.: 325 °C, drying gas: 9 L/min, nebulizer pressure: 35 psig, capillary voltage: 4000 V, fragmentor voltage: 200 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: 60–950 m/z and the acquisition rate: 1.6 spectra/s. To ensure accuracy in masses measurements, a reference mass correction was used and masses 121.050873 and 922.009798 were used as lock masses.

2.3. Forced degradation studies

Forced degradation studies were performed for the bulk substance using stock solution of toloxatone prepared in water at concentration 200 µg mL⁻¹. The working solutions were prepared by diluting the stock solutions using the proper solvent to obtain the final concentration of 10 µg mL⁻¹ and next stressed under hydrolytic, oxidative and photolytic conditions (Table 1). All the hydrolytic and oxidative degradations 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. A photostability chamber Atlas Suntest CPS+ (Linsengericht, Germany) with full UV–VIS spectrum (D65) was used as an UV–VIS source, according to ICH guidelines. The irradiance was set to 750 W/m² which corresponds to the dose of 2700 kJ/m²/h. As a UVC source a Haland HA-05 (Warsaw, Poland) ultraviolet laboratory lamp equipped with 6 W 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 toloxatone sample in a quartz cell wrapped in aluminum foil for the same period of time.

Table 1.

Stress conditions applied to toloxatone degradation.

Stress conditions	Diluting solvent	Exposure conditions	Duration (h)
Acid hydrolysis	1 M HCl	80 °C	2
Alkaline hydrolysis	0.01 M NaOH	80 °C	2
Neutral hydrolysis	H2O	80 °C	2
Oxidation	0.01% H2O2	80 °C	2
Photolysis (UV–VIS)	H2O	Room temp.	48
Photolysis (UVC)	H2O	Room temp.	2

2.4. Chemometric analysis

Three individual samples were prepared for each stressed condition as well as for not stressed control solution of toloxatone in water (STD) 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 toloxatone degradation products. The MFE parameters were optimized and the following settings were applied: maximum 1 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.

The multivariate chemometric analysis were performed under Mass Profiler Professional (MPP) software version 12.61 (Agilent and Strand Life Sciences Pvt. Ltd.). The data was normalized and aligned before the principal component analysis (PCA) was performed. This procedure allowed to evaluate qualitative differences in the registered degradation profiles of toloxatone.

2.5. In silico toxicity estimation

Acute toxicity to rodents, mutagenicity and carcinogenicity of elucidated transformation products, as well as the parent compound, were calculated with the use of following software: ACD/Percepta 14.0.0 (ACD/Labs, 2015 Release), and Vega 1.1.1. Then multivariate chemometric analysis was performed, in order to compare toxicity of the photoproducts and toxicity assessment methods. Data pre-processing and PCA analysis were performed with the use of R 3.2.3 software (GNU project). The obtained data was centered and scaled before the chemometric analysis.

3. Results and discussion

3.1. Optimization of the LC-ESI-MS/MS method

Chromatographic conditions were optimized on UHPLC reversed-phase C18 column. Various organic modifiers, buffers and elution systems including gradient elution were tested. Finally, taking into account high polarity of the parent compound as well as its transformation products, a simple isocratic elution with a mixture of acetonitrile and water (10:90, v/v) with addition of 0.1% solution of formic acid as a mobile phase was chosen. In these conditions in a quite short time of analysis (11 min) a good separation of the analyzed products was obtained (Fig. 1).

Fig. 1.

TOF extracted ion chromatograms obtained under neutral (A), acidic (B), basic (C), oxidative (D), photo UV–VIS (E) and photo UVC (F) stress conditions.

The MS conditions were optimized based on our previous study (Trawiński et al., 2017) and in the beginning electrospray ionization (ESI) was selected as a more effective ion source for this research. In TOF (MS) mode negative and positive ionization was tested and only in positive ionization all transformation products were registered. In the case of MS/MS experiments the collision energy (CID) was primarily optimized and finally for all the analyzed compounds CID was ranged from 8.7 to 20.0 eV.

3.2. Identification of forced degradation products

Under applied stress conditions eight unique degradation products (TPs) of toloxatone were formed. Most of them were characteristic for one condition, with the exception of TP1, TP2 and TP4. Two former products were common for H₂O₂ and UVC conditions. TP3 was formed only as a consequence of impact of oxidative environment. TP4, another product formed in more than one environment, was detected in both irradiated samples. Nevertheless it should be noticed that, besides of this product, TPs formation profile was dependent on applied radiation spectrum. TP5 and TP6 were formed only in UVC irradiated sample, while presence of TP7 was characteristic for UV–VIS stressed sample. TP8 was sole product of basic hydrolysis. None of the TPs was formed in acidic and neutral hydrolysis experiments.

In order to elucidate probable structures of formed TPs MS and MS/MS spectra were collected. As shown in Table 2 masses of toloxatone, as well as its transformation products were collected with good accuracy (0.09–3.79 ppm), which enabled determination of molecular formulae, and, along with information obtained from fragmentation spectra, the TPs structures.

Table 2.

Q-TOF accurate mass elemental composition and MS/MS fragmentation of the analyzed compounds.

Name	Observed in stress condition	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]+
Toloxatone	–	9.44	208.09640	208.09682	−2.02	C11H14NO3	164.10366 152.07060 145.09508 134.06004 120.08078	C10H14NO C8H10NO2 C10H12N C8H8NO C8H10N
TP1	H2O2, UVC	0.57	108.08070	108.08078	−0.74	C7H10N	93.05787 91.05297 83.05866 66.98539 65.03889	C6H7N C7H7 C5H9N C4H5N C4H3N
TP2	H2O2, UVC	1.65	224.09181	224.09173	0.36	C11H14NO4	180.10190 168.06353 136.07549 108.05501 69.06978	C10H14NO2 C8H10NO3 C8H10NO C7H8O C5H9
TP3	H2O2	1.96	240.08595	240.08665	−2.93	C11H14NO5	222.07457 206.07968 194.08350 148.07569 134.05898 122.06004 106.06482 77.03952	C11H12NO4 C11H12NO3 C10H12NO3 C9H10NO C8H8NO C7H8NO C7H8N C6H5
TP4	UVC, UV–VIS	2.54	222.07635	222.07608	1.22	C11H12NO4	178.04828 160.07337 134.05936 106.06541	C9H8NO3 C10H10NO C8H8NO C7H8N
TP5	UVC	2.75	208.09620	208.09682	−2.98	C11H14NO3	190.08630 172.07440 160.07510 144.07800 132.08000 120.07900 105.06960	C11H12NO2 C11H10NO C10H10NO C10H10N C9H10N C8H10N C8H9
TP6	UVC	5.04	208.09620	208.09682	−2.98	C11H14NO3	190.08460 72.07680 160.07440 144.07930 132.07970 120.07870 105.06830	C11H12NO2 C11H10NO C10H10NO C10H10N C9H10N C8H10N C8H9
TP7	UV–VIS	2.81	224.09171	224.09173	−0.09	C11H14NO4	206.07974 134.09441 118.06396 83.08265 69.06885	C11H12NO3 C9H12N C8H8N C6H11 C5H9
TP8	NaOH	1.01	182.11687	182.11756	−3.79	C10H16NO2	164.10613 146.09675 134.09491 120.07984 108.08078	C10H14NO C10H12N C9H12N C8H10N C7H10N

As shown in Fig. 2 fragmentation pattern of toloxatone (m/z 208.09640, C₁₁H₁₄NO₃) consisted of two paths. The first one began with elimination of a carbon dioxide molecule, which resulted in formation of m/z 160.10366 (C₁₀H₁₄NO) ion, followed by loss of water (m/z 145.09508, C₁₀H₁₂N). Elimination of propanol molecule, and formation of m/z 152.07060 (C₈H₁₀NO₂) was first stage of the second path. Then elimination of water took place, and ion of m/z 134.06004 (C₈H₈NO) was formed. Two mentioned paths eventually gave rise to formation of the most abundant ion in the spectrum – m/z 120.08078 (C₈H₁₀N).

Fig. 2.

Q-TOF MS/MS spectrum of toloxatone and corresponding fragmentation pathway.

TP1, identified as m-toluidine (m/z 108.07808, C₇H₁₀N), was formed as a consequence of the oxazolidinone ring decomposition. The most abundant peak in its MS/MS spectrum (Fig. 3) represented an ion radical (m/z 93.05787, C₆H₇N) formed after loss of a methane molecule. Then it started to gradually decompose, giving m/z 83.05866 (C₅H₉N) ion radical and products of cyclization: m/z 66.98539 (C₄H₅N) and m/z 65.03889 (C₄H₃N) ion radicals. Ion of m/z 91.05297 (C₇H₇) was a tropylium cation.

Fig. 3.

Q-TOF MS/MS spectrum and fragmentation pathway of degradation product TP1.

Accurate mass (m/z 224.09118) and corresponding molecular formula (C₁₁H₁₄NO₄) of TP2 suggested that this compound was a product of toloxatone oxidation. Its fragmentation pathway (Fig. 4) began, similarly to toloxatone, with loss of a carbon dioxide molecule (m/z 180.10190, C₁₀H₁₄NO2), and then elimination of ethanol, which resulted in formation of m/z 136.07549 (C₈H₁₀NO) ion (the most abundant peak in the spectrum). This fragmentation ion was also formed via ion of m/z 168.06353 (C₈H₁₀NO₃) which was a result of elimination of propanol from the parent molecule. Further decomposition involved loss of methanamine (m/z 108.05501, C₇H₈O) and cleavage of an aromatic ring (m/z 69.06978, C₅H₉). Based on these evidences, location of an additional hydroxyl group was restricted to an aromatic ring. Despite the data obtained from the MS/MS spectrum did not allow for unequivocal determination of its position, para relative to the oxazolidinone ring was chosen, as probably the most favored.

Fig. 4.

Q-TOF MS/MS spectrum and fragmentation pathway of degradation product TP2.

The case of TP3 (m/z 240.08595, C₁₁H₁₄NO₅) was similar to TP2, however two additional oxygen atoms were attached to the parent compound. Its fragmentation (Fig. 5) began with an unusually effortless loss of water, with formation of m/z 222.07457 (C₁₁H₁₂NO₄) ion, which suggested that one oxygen formed an N-oxide group. Then gradual decomposition of a hydroxymethyl group took place, and ions of m/z 206.07968 (C₁₁H₁₂NO₃) and 194.08350 (C₁₀H₁₂NO₃) were formed. Further stages of decomposition were similar to those observed in the case of TP2: elimination of a carbon dioxide molecule (m/z 148.07569, C₉H₁₀NO) and methyl group (m/z 134.05898, C₈H₈NO). The latter product then gave two derivatives of a tropylium cation: containing amino and hydroxyl groups (m/z 122.06004, C₇H₈NO), and dehydroxylated (m/z 106.06482, C7H8N). Therefore TP3 was probably a product of further oxidation of TP2. Ion of m/z 77.03952 (C₆H₅) was a phenyl cation.

Fig. 5.

Q-TOF MS/MS spectrum and fragmentation pathway of degradation product TP3.

TP4 (m/z 222.07635, C₁₁H₁₂NO₄), similarly to TP2, contained one additional oxygen atom. Nonetheless in this case double bond equivalent (DBE) value was lower than toloxatone (7 versus 6), which suggested presence of oxo group. Fragmentation of this product (Fig. 6) started with elimination of ethanol (m/z 178.04828, C₉H₈NO₃) or carbon dioxide along with water molecules (m/z 160.07337, C₁₀H₁₀NO). These two fragmentation ions formed then m/z 134.05936 (C₈H₈NO) ion, which then decomposed forming amino derivative of tropylium cation (m/z 106.06541, C₇H₈N).

Fig. 6.

Q-TOF MS/MS spectrum and fragmentation pathway of degradation product TP4.

TP5 (m/z 208.09620) and TP6 (m/z 208.09626) possessed the same molecular formula (C₁₁H₁₄NO₃) were both structural isomers of toloxatone. None significant differences between MS/MS spectra of these products occurred, however their fragmentation patterns did not resemble pattern of the parent compound. These observations suggested that TP5 and TP6 were almost identical products, but they remarkably differed from toloxatone. Presence of ions representing C₈H₉ formula (m/z 105.06960, Fig. 7. and 105.06830, Fig. 8) indicated that nitrogen atom in the oxazolidinone ring was replaced by a carbon atom. Therefore TP5 and TP6 were probably products of toloxatone oxazolidinone ring rearrangement. Fragmentation pathway began with loss of water molecule in both cases (m/z 190.08630 and 190.08460, C₁₁H₁₂NO₂ ions), which might suggest that the oxo group of the parent compound underwent the transformation into an alcohol group, accompanied with a migration of a double bond. Then elimination of water (m/z 172.07440 and 172.07680, C₁₁H₁₀NO) or methanol (m/z 160.07510 and 160.07440, C₁₀H₁₀NO) took place. These ions afterwards formed ions of m/z 144.07800 and 144.07930 (C₁₀H₁₀N), which underwent gradual decomposition (via C₉H₁₀N and C₈H₁₀N) forming eventually discussed C₈H₉ ion. As it was mentioned, due to lack of significant differences between fragmentation spectra of TP5 and TP6, determination of location of an additional double bond was impossible. Thus calculated logP values for two most probable structures were taken into the account. Calculations were done with the use of ALOGPS 2.1 software (VCCLAB platform, http://www.vcclab.org/). Average values of logP values were: 0.86 for imine derivative (TP5, shorter t_R), and 1.15 for enol derivative (TP6. longer t_R).

Fig. 7.

Q-TOF MS/MS spectrum and fragmentation pathway of degradation product TP5.

Fig. 8.

Q-TOF MS/MS spectrum and fragmentation pathway of degradation product TP6.

TP7 (m/z 224.09123, C₁₁H₁₄NO₃), similarly to TP2, was a product of the addition of one oxygen atom to the parent molecule. In this case it was probably attached to the oxazolidinone nitrogen atom. This assumption was based on presence of m/z 206.07974 (C₁₁H₁₂NO₃) ion, which was a product of loss of a water molecule. None of remaining fragmentation ions contained additional oxygen atom (Fig. 9). The next stage of fragmentation was elimination of methanol and cleavage of oxazolidinone ring (m/z 162.09378, C₁₀H₁₂NO ion), followed by elimination of a carbon oxide molecule (m/z 134.09441, C₉H₁₂N ion), and further decomposition (ion of m/z 118.06396, C₈H₈N). Two low mass ions (m/z 83.082365 and 69.06885) represented residuals of an aromatic ring (C₆H₁₁ and C₅H₉ respectively).

Fig. 9.

Q-TOF MS/MS spectrum and fragmentation pathway of degradation product TP7.

Accurate mass and corresponding molecular formula of TP8 (m/z 182.11662 and C₁₀H₁₆NO₂ respectively) suggested that loss of carbon oxide took place in case of this product. This assumption was supported by its fragmentation spectrum (Fig. 10), consisting of four main peaks, representing fragments similar to those observed in the case of toloxatone. Firstly elimination of a water molecule took place, and ion of m/z 164.10613 (C₁₀H₁₄NO) was formed. Then next water molecule was eliminated, which resulted in formation of m/z 146.09675 (C₁₀H₁₂N) ion, and was followed by gradual decomposition of propenyl chain (ions of m/z 134.09491, C₉H₁₂N and 120.07984, C₈H₁₀N). The last stage was formation of m-toluidine (m/z 108.08078, C₇H₁₀N).

Fig. 10.

Q-TOF MS/MS spectrum and fragmentation pathway of degradation product TP8.

The suggested transformation pathway of toloxatone in the investigated stress conditions was shown in Fig. 11.

Fig. 11.

Forced degradation pathway of toloxatone in tested conditions.

3.3. Chemometric study

In order to perform the multivariate chemometric analysis all the obtained chromatographic profiles (21 chromatograms) registered in TOF (MS) mode were aligned with MPP software giving 62 entities. After a build-in MPP filtration including sample abundance, setting the fold change (FC) threshold on the level not less than 4 and one-way ANOVA statistical test (p = 0.05), 31 entities were finally selected for the chemometric study. The PCA analysis based on this data showed a visible categorization of all the analyzed groups of the forced degradation samples (Fig. 12). Basic (NaOH) stressed samples stood out from the other samples while neutral (H₂O) and acidic (HCl) stressed samples were very close to each other and to control samples without degradation (STD). It should be also noticed that the samples subjected to UVC irradiation visibly stood out from the ICH photolytic conditions (UV–VIS) and are placed between oxidative (H₂O₂) and UV–VIS stressed samples. In the presented PCA analysis the first three components (PC) explained 70.7% of the total variance.

Fig. 12.

The 3D PCA plot of forced degradation profiles of toloxatone in tested conditions.

3.4. Computational estimation of toxicity

In order to estimate and compare toxicity of toloxatone as well as its TPs, carcinogenic and mutagenic potential, and acute toxicity to rodents was calculated.

Probability of carcinogenic activity was estimated with the use of CAESAR 2.1.9 model provided by Vega software (version 1.1.1). According to this model, most of identified compounds possessed lower carcinogenic potential than the parent compound (probability equaled 0.71, Supplementary data – Table S1). Higher potential was predicted only for one compound (TP5).

Mutagenicity (probability of positive Ames test) was predicted by model included in Percepta software (ACD/Labs). Similarly as in the case of carcinogenicity, majority of TPs was marked by lower mutagenic potential than toloxatone (probability equaled 0.33). Only two compounds – TP1 and TP5 – possessed higher mutagenic potential. Nevertheless even in their case the probability did not exceed 0.5 (Supplementary data – Table S2).

Percepta software was also used to estimate acute toxicity to rodents. In this case six models were applied, four for mice (intraperitoneal, intravenous, subcutaneous and oral), and two for rats (intraperitoneal and oral). In order to facilitate interpretation of calculated results (Supplementary data – Table S3), principal component analysis of obtained matrix was performed. This chemometric tool enables reduction of data dimensionality, and visualization of relationships between samples (compounds) as well as variables (toxicity endpoints). As was shown in Fig. 13A beside two pairs, Mouse Oral – Rat Oral and Mouse Intravenous – Mouse Subcutaneous, variables were rather weakly correlated. Variables representing Rat Intraperitoneal and Mouse Subcutaneous toxicity were almost orthogonal (cos θ close to 0). Points representing TP2 and TP4 were plotted close to toloxatone, which corresponded to their similar properties. TP6 and TP8 were less toxic according to all of the applied models (variables values increase in parallel with LD₅₀). Three compounds placed in upper-right part of the plot (TP1, TP3 and TP7) were more toxic that the parent compound. TP5, the most outlying compound, was less toxic according to Mouse Intravenous and Mouse Subcutaneous models, but more toxic when other models were taken into account.

Fig. 13.

Comparison of toxicity of forced degradation products by PCA: acute toxicity to rodents, OR – Oral, IV – Intravenous, S.C. – Subcutaneous, IP –Intraperitoneal (A); overall toxicity (B).

The overall toxicity was estimated by performing of PCA on set of three variables, one from each toxicity category. As it can be seen form Fig. 13B TP6 was very similar to the parent compound (high carcinogenic potential, low toxicity to rodents and moderate mutagenicity). Amongst two outlying products, TP5 was highly mutagenic and carcinogenic, while its toxicity to mice was low. The second outlier, TP1, possessed moderate mutagenic and carcinogenic potential, and very low toxicity to mice. Most TPs were placed in upper part of the plot, which corresponded to their low mutagenic and carcinogenic potential, and low to moderate toxicity to mice.

4. Conclusion

The degradation behavior of toloxatone under hydrolytic – acid, base and neutral, oxidative and photolytic – as per ICH guidelines and UVC stress was studied. It was observed that the tested antidepressant drug is fragile towards basic hydrolysis, oxidative conditions and UVC irradiation. Eight degradation products were found and based on MS/MS fragmentation spectra their structural elucidation was performed. The most effective degradation processes in terms of the number of transformation products was UVC photolysis and five TPs (TP1, TP2, TP4, TP5, TP6) were found in this case. Two of them (TP5 and TP6) were formed only in these conditions. The other three identified transformation products are characteristic for sole degradation conditions – oxidative (TP3), base (TP8) and UV–VIS photolytic (TP7).

The multivariate chemometric analysis (PCA) allowed effortless characterization of the registered degradation profiles which can be a useful method for a fast preliminary degradation study of drugs.

Additionally, the toxicity of the characterized degradation products was estimated with the use of in silico methods and compared with multivariate chemometric method. Two of these products (TP1 and TP5) were found as more toxic that the parent compound and the other transformation products.