Journal of Pharmaceutical and Biomedical Analysis 96 (2014) 58–67

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Degradation pathways study of the natriuretic and ␤-adrenoceptor antagonist tienoxolol using liquid chromatography–electrospray ionization multistage mass spectrometry Ines Gana a,f,1 , Annabelle Dugay a,1 , Théo Henriet b,c , Ivo B. Rietveld a , Mélisande Bernard c , Christophe Guechot d , Jean-Marie Teulon e , Fathi Safta f , Najet Yagoubi b , René Céolin a , Bernard Do b,c,∗ a

University of Paris-Descartes, Faculty of Pharmacy, Laboratory of Physical Chemistry, 4 avenue de l’Observatoire, 75006 Paris, France University of Paris-Sud, Faculty of Pharmacy, Groupe Matériaux et Santé EA 401, 5 rue Jean-Baptiste Clément, 92296 Châtenay-Malabry, France c Assistance Publique-Hôpitaux de Paris, Agence Générale des Equipements et Produits de Santé, Département de Contrôle Qualité et Développement Analytique, 7 rue du Fer à Moulin, 75005 Paris, France d Solvay SA, 25 rue de Clichy, 75009 Paris, France e 78170 La Celle St Cloud, France f University of Monastir, Faculty of Pharmacy, Laboratory of Analytical Chemistry, rue Ibn Sian, 5000 Monastir, Tunisia b

a r t i c l e

i n f o

Article history: Received 11 January 2014 Received in revised form 5 March 2014 Accepted 11 March 2014 Available online 25 March 2014 Keywords: Tienoxolol Degradation pathways Liquid chromatography–mass spectrometry Kinetics Active pharmaceutical ingredient

a b s t r a c t Tienoxolol is a pharmacologically active molecule designed with the functional groups ketothiophene, alkyl benzoate and arylpropanolamine so as to combine a diuretic and a ␤-adrenoreceptor antagonist into a single molecule. Its degradation products generated in several stress media have been determined by high-pressure liquid chromatography (HPLC) coupled to a hybrid mass spectrometer with a triple quadrupole-linear trap. A Polaris® column with a C18-A stationary phase and a linear gradient mobile phase composed of a mixture of trifluoroacetic acid 1% (v/v) and acetonitrile allowed for optimal separation. Structural elucidation of the degradation products has been based on MS/MS techniques, by comparing their fragmentation patterns to the precursor’s data. Up to seven degradation products of the active ingredient, resulting from hydrolysis, oxidation, dehydration and transamidation have been identified, covering a range of possible degradation pathways for derivatives with such functional groups. Kinetics have been studied to assess the molecule’s shelf life and to identify the most important degradation factor. © 2014 Elsevier B.V. All rights reserved.

1. Introduction In the 1980s, one of the drug development strategies was to associate two active moieties in a single molecule to facilitate common therapeutic approaches for treating hypertension. Tienoxolol is a molecule with multiple functional groups (ethyl 2-[3-[(1,1-dimethylethyl) amino]-2-hydroxypropoxy]-5[(2-thienylcarbonyl) amino] benzoate), so as to combine both natriuretic and ␤-adrenoceptor antagonist effects. Its pharmacological activities had previously been demonstrated not only in

∗ Corresponding author at: University of Paris-Descartes, Faculty of Pharmacy, Département de Physico-Chimie du Médicament, Laboratory of Analytical Chemistry, 4 avenue de l’Observatoire, 75006 Paris, France. Tel.: +33 662306275. E-mail address: [email protected] (B. Do). 1 Contributed equally to this study and are therefore considered as first authors. http://dx.doi.org/10.1016/j.jpba.2014.03.016 0731-7085/© 2014 Elsevier B.V. All rights reserved.

normotensive conscious rats and monkeys after oral dosing, but also in humans [1]. In healthy volunteers, the active ingredient behaved as an early acting and relatively long-lasting selective beta1 -adrenoceptor blocking drug endowed with significant natriuretic properties [2]. Our previous work concerning the solid-state of tienoxolol demonstrated that hydrolysis had occurred during storage protected from light at room temperature [3]. A literature survey, however, did not reveal any further information about the stability profile of tienoxolol or its potential degradation products likely to form in time and/or under stress conditions. In addition, this study contributes to the knowledge about the stability of compounds containing the functional groups aryl keto thiophene, benzoate and/or arylpropanolamine. In this paper, we have focused on the identification and the characterization of degradation products generated in solution. To this end, high performance liquid chromatography coupled with

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multistage mass spectrometry (HPLC–MSn ) was used. In accordance to ICH guidelines [4], different types of media were applied in order to simulate the rate of degradation of active pharmaceutical substances, where degradation can occur along many pathways such as basic and acidic hydrolysis, oxidation, photo-degradation or temperature. The proposed structures for the observed degradation products and their formation pathways were compared with previously published relevant data. In addition, quantitative determinations of tienoxolol in the presence of its degradation products have provided kinetic data with respect to specific stress conditions.

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the Q-Pod Milli-Q system (Millipore, Molsheim, France), were used for dissolution. Stock standard solution (600 ␮g mL−1 ) was prepared by dissolving 120 mg of the compound in 200 mL of a mixture of ultrapure water/acetonitrile 20/80 (v/v). A working solution was prepared from this stock solution by dilution in ultrapure water in order to obtain a final concentration of 300 ␮g mL−1 . Hydrogen peroxide 30% was purchased from Carlo Erba SDS (Val de Reuil, France). Sodium hydroxide 32%, obtained from VWR international Prolabo (Fontenay sous bois, France) and hydrochloric acid 32%, obtained from Merck (Fontenay sous Bois, France), were both used at 1 M. Technical TFA (99%) was purchased from VWR International Prolabo (Fontenay sous bois, France).

2. Materials and methods 2.3. Degradation protocol 2.1. Instrumentation A reversed-phase liquid chromatography (LC) method with mass spectrometry (MS) detection was set up to study the impurity profile of Tienoxolol. The LC system (Dionex, Les Ulis, France) consists of a quaternary pump, a vacuum degasser and an autosampler and is piloted by Chromeleon® software version 6.80 SR11 (Dionex, Les Ulis, France). The selected analytical column was a Varian Polaris C18-A (P/N A2000150X020, Agilent Technologies, Santa Clara, CA, USA), 150 mm length, 2.0 mm internal diameter and 5 ␮m particle size. The flow rate was set at 1.0 mL min−1 and the sample injection volume was 20 ␮L. A mobile phase linear gradient was set up (solvent A: TFA in ultrapure water 0.1% (v/v), solvent B: acetonitrile) in order to prevent co-elution of impurities and to take into account late eluting impurities (Table 1). The chromatographic effluent stream was directed into the mass spectrometer (MS) interface, which consisted of an electrospray ionization (ESI) source. Mass spectrometry analyses were carried out with a triple quadrupole linear ion trap HybridQtrap 3200 MS system (AB Sciex, Framingham, USA). The MS operated in the positive ionization mode with the following conditions: ion spray voltage was set at 5.5 kV, curtain gas (N2 ) flow rate was set at 30 psi, nebulizer gas (air) flow rate was set at 40 psi, heater gas (Air) flow rate was set at 50 psi, temperature was set at 600 ◦ C, Q1 entrance potential was 56 V in scan mode. Nitrogen was used as a damping and collision gas. For MSn experiments the ions of interest were isolated in the ion trap with an isolation width of 3 Da and activated at different collision energy levels (CEL) to find the optimum conditions for a distinct fragmentation. A CEL of 30% optimally generated the highest intensity of the precursor ion needed for further CID experiments. Different chemical structures were elucidated using three different types of scanning modes (EMS+, ER+ and EPI+). The MS data were treated with Lightsight® (AB Sciex, Les Ulis, France) and ACD/Labs MS manager® (ACD/labs, Strasbourg, France) softwares. 2.2. Materials and reagents Tienoxolol (MW: 420.51 g mol−1 ) samples were provided by UPSA Pharma (Agen, France) and stored in the dark at room temperature. Analytical grade acetonitrile, purchased from Sigma–Aldrich (St Quentin-Fallavier, France) and ultrapure water, obtained from Table 1 HPLC gradient program. Temps (min)

Solvent A: trifluoracetic acid in ultrapure water 0.1% (v/v)

Solvent B: acetonitrile

0 → 25 25 → 40 40 → 55 55 → 58

75 75 → 30 30 30 → 75

25 25 → 70 70 70 → 25

Hydrolytic stress studies using HCl 0.05 M and NaOH 0.05 M as treatments were carried out as follows: (a) in acidic condition, 1 mL HCl (0.1 M) was added to 1 mL of TXL stock solution and placed at 80 ◦ C for 2.5 h. This solution was then neutralized by addition of 1 mL NaOH (0.1 M) and placed at room temperature; (b) in alkaline condition, 1 mL NaOH (0.1 M) was added to 1 mL TXL stock solution and placed at 80 ◦ C for 2.5 h. This solution was then neutralized by addition of 1 mL of HCl (0.1 M) at room temperature; and (c) at unmodified pH, 1 mL of ultrapure water was added to 1 mL of TXL stock solution and placed at 80 ◦ C for 4 h. Oxidative stress studies using hydrogen peroxide 15% (v/v) as treatment was carried out as follows: 1 mL H2 O2 (30%, v/v) was added to 1 mL of TXL stock solution and left at 25 ◦ C for 24 h. A 5 mL glass vial containing 1 mL of TXL working solution was exposed to light at a spectral wavelength of 254 nm and an intensity of 200 W h m−2 as part of the photo-degradation studies. A Universal UV lamp, CAMAG TL-900 (Muttenz, Suisse), with two variable wavelengths (254 and 350 nm) was used to perform this treatment. 3. Results and discussion 3.1. Optimization of chromatographic conditions and method performance The LC–UV–MSn method was used to characterize TXL degradation products generated under stress conditions. HPLC was used to separate TXL and its degradation products in a single run. In acidic media, TXL and the degradation products still carrying an amine function are charged by protonation and thus can form ion pairs with TFA. A gradient mode for the mobile phase was used to improve the resolution between the different compounds, i.e. tienoxolol and its degradation products. Representative LC/MS chromatograms of samples that underwent the different stress conditions described above are shown in Fig. 1. Most of the degradation products eluted before TXL and so, may be more hydrophilic than the parent compound (Table 2). Only one degradation product showed a higher retention time than TXL. The extracted chromatograms of ions from the eluted compounds revealed the presence of seven degradation products. Among them, the product eluted at RT 4.8 min and subsequently designated as DP1 , had already been observed before in solutions of untreated TXL. Chromatographic parameters of tienoxolol and its degradation products resulting from different media used for the stress tests TXL were well resolved from its degradation products for all degradation conditions (Table 2). A component detection algorithm (CODA) analysis also allowed examining the main peak purity, showing that it uniquely contained signals from TXL and the solvents regardless of the sample. The regression analysis using a linear model expressing TXL concentrations as a function of MS signals within a range 15–150 ␮g mL−1 , resulted in a determination coefficient

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Fig. 1. Extracted ion chromatograms of protonated molecules of TXL and its degradation products.

R2 of 0.9987 and a y-intercept of the linear equation which was statistically insignificant (p = 0.294). The distribution of the residuals can well be approximated with a normal distribution according to the p-value of the Shapiro–Wilk normality test (p = 0.198), so that it could be safely assumed that the calibration data fitted to a linear model. The LOD and LOQ values based on the standard error of the residuals and the slope of the calibration plot were 3.4 and 10 ␮g mL−1 , respectively. The repeatability verified by a sixfold analysis of the concentration level 100 ␮g mL−1 yielded a RSD

inferior to 2%, and the intermediate precision studied over three different days following the same protocol, led to a RSD equal to 2.05%. 3.2. Degradation of tienoxolol TXL mainly degraded to one or two degradation products in most of the stress conditions, except in oxidizing media, which produced a total of five degradation products (Table 2 and Fig. 1).

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Table 2 Chromatographic parameters of tienoxolol and its degradation products resulting from different media used for the stress tests.

RT (min) Oxidation Acidic Basic Temperature UV–vis Average RT Average peak efficacy Average resolution factors

DP1

DP2

DP3

DP4

DP5

DP6

Tienoxolol

DP7

4.80 4.79 4.79 4.85 4.84 4.81 200 0.4

5.11 – – 5.10 – 5.11 14,457 2.7

– – – 6.03 6.01 6.02 2229 5.9

10.03 – – – – 10.03 2228 1.1

11.38 – – – – 11.39 887 1

12.81 – – – – 12.81 1420 3.9

16.84 16.78 16.79 16.89 16.87 16.83 9801 41.3

– 34.40 – – – 34.40 655,147

Fig. 2. 1/C = f(t) graphs.

In accordance with their elution order, degradation products were labeled DP1 to DP7 . The correlation between the decrease of TXL signal and the increase of degradation products signals was assessed by calculation of the Pearson correlation coefficients.

lower under this condition than after 7 days of exposure to oxidative conditions. Similar observations were made regarding DP3 . 3.3. Kinetics

3.2.1. Influence of acidic–basic media The influence of the acidic and basic media was assessed over a maximum observation time of 7 days (in HCl 0.05 M and NaOH 0.05 M). Acidic and basic media caused an increase in DP1 , already present in the original sample as stated above. Acidic media yielded DP7 with a Pearson correlation coefficient of −0.76, whereas no extra peaks were observed in basic media. TXL was very susceptible toward alkaline stress with a nearly complete conversion into DP1 after exposure for 7 days in NaOH 0.05 M. 3.2.2. Influence of oxidation media Fifteen percent (v/v) H2 O2 caused TXL to degrade for up to 50% after a seven-day period with the appearance of DP2 , DP4 , DP5 , and DP6 in addition to DP1 . DP2 , DP4 , and DP6 arose quickly, whereas DP5 was produced more gradually. The Pearson correlation coefficients of R = −0.76 (DP2 ), R = −0.73 (DP4 ), R = −0.67 (DP5 ) and R = −0.80 (DP6 ) confirm that the decrease in tienoxolol was correlated with the formation of these degradation products. 3.2.3. Influence of light Photo-degradation was performed at room temperature and humidity. The percentage of TXL exposed to light radiation decreased down to 70% after 7 days. DP3 was found with a Pearson correlation coefficient of −0.82. 3.2.4. Influence of temperature At 80 ◦ C DP1 increased and DP2 and DP3 were also produced (R = −0.67, R = −0.81, R = −0.74). However DP2 content was

Tienoxolol decreased as a function of time, regardless of the stress conditions considered. The results were plotted as 1/C = f(t) profiles for the different conditions (Fig. 2). Reaction rate constants (k) possess the same order of magnitude for the decomposition by temperature and in acidic medium, whereas k doubled in the presence of the oxidizing agent. Therefore, the time at which 50% of the substance is lost depends on the stress condition tested and obviously, TXL is more susceptible to oxidation than hydrolysis in acidic medium (Table 3). 3.4. Characterization The protonated TXL molecule [M+H]+ was observed at m/z 421 (data not shown). The ESI-MS2 spectrum of protonated TXL exhibited several major product ions at m/z 365, 337, 319, 301, 291, 264, 246, 130, 111, and 74. A number of the TXL product ions are

Table 3 Kinetic results of tienoxolol under the investigated stress conditions. Stress conditions

Rate constant (day−1 )

T0.5 (day)

Correlation coefficient r2

Temperature Oxidative medium Acidic medium UV–vis

0.0006 0.0015 0.0007 0.0010

18.5 7.4 15.9 11.1

0.9417 0.9065 0.9741 0.9170

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Fig. 3. Proposed ESI-MSn fragmentation pathways of protonated TXL.

very likely formed by protonation of the amine nitrogen. However, there are also product ions that are more readily explained by dissociation of an ether oxygen-protonated and an ester oxygenprotonated species [5]. In first instance, the discussion will focus on the product ions most likely originating from the amine nitrogen-protonated species. The first is caused by a neutral loss of a 56 Da moiety corresponding to CH2 C(CH3 )2 , which is a consequence of the Nde-alkylation process or the N C bond cleavage as illustrated in Fig. 3. In turn, this product ion might undergo two plausible fragmentation pathways facilitated by a hydrogen transfer from the amine to the ester carboxylic oxygen. Hence, the product ion at m/z 365 could either lose ethanol (46 Da) by a mechanism involving an

intramolecular four-center rearrangement leading to the formation of an acylium ion at m/z 319 by ␣ cleavage, or lose ethylene justified by the presence of the ion at m/z 337 [6]. The precursor ion at m/z 319 fragmented into product ions at m/z 291, 301 and 246, indicating departures of carbon monoxide [7], water and hydroxyl amino propene residue (73 Da), respectively by intramolecular four and six-center rearrangements. The product ion at m/z 218 could derive from the m/z 246 precursor by elimination of CO, then through an intramolecular rearrangement, as previously described by March and Miao [8]. Ions at m/z 111 and 85 could be produced by a pseudo-molecular ion after protonation of the amide nitrogen keto-thiophenone [5] (Fig. 3). The most abundant product ions resulting from the

Fig. 4. Proposed ESI-MSn fragmentation pathways of protonated ion at m/z 365.

Table 4 Major product ions and proposed structures of the degradation products. m/z MH+ (DP)

m/z Products ions

m/z MH+ (DP) 341 (DP5 )

285 268 239 221 166 74

365 (DP2 )

337 319 301 291 264 246 218 111 85 74 309

288 (DP6 )

260

375 (DP3 )

263 234 190 136 319

531 (DP7 )

217 202 176 111 475

393 (DP1 )

Proposed structure

246 (DP4 )

m/z Products ions

429 411 301 264 246 184 164 111 85

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301 264 246 218 111

Proposed structure

240 194 176 133 121 74

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Fig. 5. Proposed ESI-MSn fragmentation pathways of protonated ion at m/z 288.

tienoxolol ether oxygen-protonated ion are m/z 130 and 74, arising from neutral loss of the substituted phenol part (Fig. 3). The difference between m/z 130 and 74 was due to the N-tertio-butyl residue. The peak at m/z 74 is characteristic of the propanolamine residue from the side chain [9]. Degradation product DP1 , with the shortest retention time of 4.8 min, is the most polar of the observed degradation products. The corresponding pseudo-molecular ion [M+H]+ at m/z 393 yielded several major product ions at m/z 337, 319, 301, 264, 246, 218, and 111. A thorough analysis of the fragmentation scheme of DP1 demonstrates that it completely reflects the fragmentation pathway of TXL. A characteristic loss of a 56 Da moiety from the precursor ion at m/z 393 as well as successive departures of a 73 Da moiety, water molecules, and carbon monoxide, were observed. Nevertheless, the neutral loss of a 46 Da moiety, previously

considered as specific to the acetic ester group of tienoxolol, was absent from the CID MS spectrum of DP1 . This difference compared to the tienoxolol CID MS spectrum, allowed the identification of the degradation product DP1 as a compound formed by the hydrolysis of the ester function [5] (Table 4). Besides, a difference in molecular mass of 28 Da compared to tienoxolol supports this hypothesis. In addition, the fact that the ester function had transformed into a carboxylic acid was also demonstrated by the rapid elution of DP1 from the stationary phase, owing to a lower hydrophobicity. As stated above, this degradation product had already been found, but to a lesser extent, in freshly prepared solutions (data not shown) suggesting that the TXL sample was not pure from the start and/or had degraded during its storage. With a retention time of 5.1 min, degradation product DP2 is one of the fastest eluting compounds. It is hardly separated from DP1 and is detected at m/z 365.The CID MS spectrum indicates the presence of six major product ions at m/z 309, 263, 245, 234, 190 and 136. It is worth noting that the characteristic ion of the propanolamine residue at m/z 74 was still present. The acylium ion at m/z 111 that reflects the presence of the keto-thiophene group was not detected in the CID MS spectrum, suggesting that a transformation had occurred in this part of the precursor. A difference in molecular mass of 56 Da compared to protonated tienoxolol might in first instance point to a product formed by N-dealkylation of tienoxolol. However, this assumption could not be retained owing to the detection of the m/z 365 → 309 transition corresponding to the departure of a 56 Da moiety, confirming the presence of the N-tertio-butyl residue in the structure of DP2 . Besides, the loss of a 46 Da moiety due to the m/z 309 → 263 transition, demonstrates that the ester function was not impacted

Fig. 6. Proposed ESI-MSn fragmentation pathways of protonated ion at m/z 531.

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by the change. As a consequence, the absence of m/z 74 suggests that the propanolamine residue was lost by degradation to yield DP2 . As stated above, the absence of the acylium ion at m/z 111 demonstrates unambiguously the replacement of the ketothiophene group by another one, which could be an ␣-keto-acid (Table 4). Based on these assumptions, the fragmentation pathway proposed in Fig. 4, turns out to fit with the data extracted from the mass spectrum. As shown in Fig. 4, the product ions at m/z 234 may arise from the product ion at m/z 263 by intra-molecular rearrangement followed by cyclization and formation of an imine. With a four-center rearrangement mechanism, the loss of a 29 Da moiety was observed, corresponding to the departure of CH2 NH. From there, hydrogen rearrangement and elimination of CO2 gave rise to the product ion at m/z 190. Thereafter, successive losses of carbon monoxide and acetylene generated the product ion at m/z 136. Degradation product DP3 had an elution time of 6 min and it was detected at m/z 375, thus showing a mass shift of 18 Da from the protonated ion of DP1 . This leads to the hypothesis of a secondary degradation product formed by dehydration of DP1 (Table 4), which

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was further corroborated by similarities observed between the CID MS spectra regarding the common mass transitions and product ions. Degradation product DP4 had an elution time of 10 min and was characterized by a protonated ion at m/z 296. Similar to DP2 , the acylium ion at m/z 111 was not observed, marking the absence of the keto-thiophene group from the molecule. Based on the nitrogen rule, the protonated ion should be composed of an odd number of nitrogen functions. Considering the fact that characteristic losses of 56 Da and 46 Da are still observed in the CID MS spectrum, one can expect that the secondary amine function remains, whereas the aromatic amide function is absent from the structure of DP4 (Table 4). Degradation product DP5 had a retention time of 11.4 min and exhibited a pseudo-molecular ion at m/z 341. According to the nitrogen rule, the molecule would contain an even number of nitrogen functions. The MS2 spectrum of m/z 341 exhibits five major product ions at m/z 285, 268, 239, 221 and 166. Once more, the product ion characteristic of the keto-thiophene moiety at m/z 111 was entirely absent from the CID MS spectrum of DP5 . This sug-

Fig. 7. Proposed reaction pathways to account for the formation of the degradation products DP1 to DP7 .

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gests that the change only occurs at this part of the precursor. Previously observed losses, such as 56 Da and 46 Da, and the characteristic product ion at m/z 74 were still observed. Therefore one may assume that the ester function and the ether side chain have remained intact. Hence, the postulated structure of the degradation product could be constructed by introducing an N-aryl methanolamine residue instead of the keto-thiophene group; this would be in accordance with the resulting molecular mass of DP5 (Table 4). Degradation product DP6 was one of the slowest eluting compounds, exhibiting a retention time of 12.8 min, whereas its pseudo-molecular ion was detected at only m/z 288. Subjected to the same CID conditions as the other elution compounds, the mass spectrum is poorer in structural information. The MS data exhibit an intense characteristic peak of the keto-thiophene moiety at m/z 111 and 85, but no sign related to the ether-propanolamine side chain was detected. Losses related to the N-tertio-butyl and arylester functions are also absent (Table 4). The product ion at m/z 260 could derive from the precursor by elimination of CO and the ion at m/z 202 could be formed by the successive departures of CO2 and C2 H2 O from the pseudo-molecular ion. Then through intra-molecular rearrangements, the ion at m/z 176 was obtained after a loss of an acetylene function. Based on the m/z value of the protonated ion of 288 Da, and in accordance with product ions at m/z 260, 202, 176, 111 and 85 and the tienoxolol structure, a structure has been proposed for DP6 , although it may require further supporting evidence (Table 4). A proposition for the MS fragmentation pathways can be found in Fig. 5. Degradation product DP7 with a protonated ion at m/z 531 has to be far more hydrophobic than TXL because of its high retention time observed at 34.5 min. This chromatographic behavior was attributed to its higher molecular mass and the presence of weakly polar functions. The MS2 mass spectrum revealed several major product ions and some of them were formed by characteristic neutral losses of 46 Da, 55 Da and 56 Da moieties, also observed for TXL. The other losses appeared to be specific to DP7 . The difference of 110 Da in molecular mass between DP7 and TXL could be generated by an addition of an alketo-thiophene unit. The acylium ion at m/z 111 appeared to be one of the most intense product ions in the CID mass spectrum. As proposed in Fig. 6, a neutral loss of the 110 Da moiety is clearly demonstrated, whereas it has not been detected for some of the other degradation compounds. The m/z 411 → 301 transition is a clear supporting element. Judging from the presence of the product ion at m/z 184, another keto-thiophene unit may be connected to the secondary amine function to form an aliphatic amide function (Table 4). 3.5. Proposed degradation pathways of Tienoxolol From the MS chromatograms of degraded tienoxolol, a total of seven degradation products were observed. Some of these degradation products can be explained by hydrolysis of the ester function [10] or by dehydration of the alcohol function present on the etherpropanolamine side chain, which may be initiated in acidic media. DP7 is most likely the results of a transamidation reaction, involving the transfer of the keto-thiophene moiety from the aromatic amide to the secondary amine (Fig. 7). In solution, the ␤-blocking part of the API, combining the aminopropanol group and the benzyl, appears to be reactive under stress conditions. This finding is consistent with what has been reported for notably propranolol, a ␤-adrenergic antagonist [11]. Other degradation products can result from oxidation in the presence of hydrogen peroxide, affecting the amino keto-thiopene moiety. The oxidation reactions lead to the formation of DP2 , DP4 and DP5 as depicted in Fig. 7. DP2 may result from oxidation of

the thiophene moiety giving rise to the formation of an ␣-keto acid, as has been reported previously for tiabagine, an antiepileptic agent, in the presence of hydrogen peroxide [12]. The same reaction followed by decarboxylation could explain the existence of DP5 . With the present results, it is not possible to propose a mechanism for the formation of DP6 . The CID of the protonated degradation product generated few product ions and further experiments, like NMR analysis, may be necessary to confirm the proposed structure. 4. Conclusion An LC method has been developed for the analysis of tienoxolol and its degradation products, as part of a study on its stability profile under stress conditions. LC/MSn proved to be a powerful tool in the identification of the degradation products of tienoxolol for which seven impurities were characterized. Some fragmentation mechanisms arise from the nitrogenprotonated ions and the others from the ether-oxygen protonated ions and ester-oxygen protonated ions. Diagnostic product ions have been identified and used as markers, i.e. the ion at m/z 74, characteristic of the propanolamine moiety and the ion at m/z 111 specific to the keto-thiophene group. In the same manner, loss of 56 Da was assigned to the tertio-butyl function from the ether-side chain and 46 Da to ethanol from the aryl-ester group. Most of the degradation products could be explained by hydrolysis of the benzoate group, dehydration affecting the ether-side chain of the ␤-adrenergic moiety and oxidation of both ketothiophene (which is part of the diuretic moiety) and propanolamine groups, Some intermolecular interactions have also been highlighted in acidic media involving a transamidation reaction, which resulted from the attachment of ketothiophene to the tertiary amine function. But apart from hydrolysis, kinetic studies have clearly identified oxidative conditions as the most influencing factor of degradation. These results can provide indications for degradation pathways of other compounds with similar structures or the same functional groups. References [1] E. Bouley, J.M. Teulon, M. Cazes, A. Cloarec, R. Deghenghi, p(Thienylcarboxamido)phenoxy propanolamine derivatives as diuretic and beta-adrenergic receptor blocking agents, J. Med. Chem. 29 (1986) 100–103. [2] A. Berdeaux, E. Loueslati, J.L. Gerard, E. Pussard, J.F. Giudicelli, Evaluation of the natriuretic and beta-adrenoceptor-blocking effects of tienoxolol in normal volunteers, Fund. Clin. Pharmacol. 5 (1988) 441–454. [3] N. Mahé, B. Do, B. Nicolaï, I.B. Rietveld, M. Barrio, J.L. Tamarit, R. Céolin, C. Guéchot, J.M. Teulon, Crystal structure and solid-state studies of aged samples of tienoxolol, an API designed against hypertension, Int. J. Pharm. 422 (2012) 47–51. [4] Guidance for Industry Q1A(R2) Stability Testing of New Drug Substances and Products, in: International Conference on Harmonisation of Technical Requirement for Registration of Pharmaceuticals for Human Use, March, 2003. [5] L. Alana, Upthagrove, Murray Hackett, Wendel L. Nelson, Mass spectral fragmentation pathways of propranolol related B-fluorinated amines studied by electrospray and electron impact ionization, Rapid Commun. Mass Spectrom. 13 (1999) 1671–1679. [6] K. Tóth, L. Nagy, A. Mándi, A. Kuki, M. Mézes, M. Zsuga, S. Kéki, CollisionInduced dissociation of aflatoxins, Rapid Commun. Mass Spectrom. 27 (2013) 553–559. [7] R. Vessecchi, F.S. Emery, N.P. Lopes, S.E. Galembeck, Electronic structure and gas-phase chemistry of protonated A- and B-quinonoid compounds: a mass spectrometry and computational study: study of protonated A- and Bquinonoid compounds, Rapid Commun. Mass Spectrom. 27 (2013) 816–824. [8] R.E. March, X.-S. Miao, A fragmentation study of kaempferol using electrospray quadrupole time-of-flight mass spectrometry at high mass resolution, Int. J. Mass Spectrom. 231 (2004) 157–167. [9] M.H. Pham, M.C. Menet, A. Dugay, A. Regazzetti, D. Dauzonne, N. Auzeil, D. Scherman, G.G. Chabot, Characterization of monohydroxylated derivatives of the anticancer agent flavone-8-acetic acid by liquid chromatography with online UV and mass spectrometry, Rapid Commun. Mass Spectrom. 21 (2007) 3373–3386.

I. Gana et al. / Journal of Pharmaceutical and Biomedical Analysis 96 (2014) 58–67 [10] C. Guechot, A. Bertrand, P. Cramaille, J.M. Teulon, Analytical profile of the new diuretic beta-blocking agent tienoxolol hydrochloride, Arzneimittel-Forschung 38 (1988) 655–660. [11] E. Isarain-Chávez, R.M. Rodríguez, P.L. Cabot, F. Centellas, C. Arias, J.A. Garrido, E. Brillas, Degradation of pharmaceutical beta-blockers by electrochemical advanced oxidation processes using a flow plant with a solar compound parabolic collector, Water Res. 45 (2011) 4119–4130.

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[12] M. Hubert-Roux, M. Skiba, A. Sughir, M. Lahiani-Skiba, F. Olivier-Chanu, V. Levacher, C.M. Lange, Identification of tiagabine degradation products using liquid chromatography with electrospray ionization multistage mass spectrometry and ultra-performance liquid chromatography/high-resolution mass spectrometry: identification of tiagabine degradation products, Rapid Commun. Mass Spectrom. 26 (2012) 287–296.

Degradation pathways study of the natriuretic and β-adrenoceptor antagonist tienoxolol using liquid chromatography-electrospray ionization multistage mass spectrometry.

Tienoxolol is a pharmacologically active molecule designed with the functional groups ketothiophene, alkyl benzoate and arylpropanolamine so as to com...
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