Philippe-Henri Secrétan†
a,
Hassane Sadou-Yayé†ab,
Caroline Aymes-Chodura,
Mélisande Bernardac,
Audrey Solgadid,
Fatma Amranic,
Najet Yagoubia and
Bernard Do*ace
aUniversité Paris Sud, UFR de Pharmacie, Groupe Matériaux et Santé, Institut d'Innovation Thérapeutique, 5, rue Jean Baptiste Clément, 92296 Châtenay-Malabry, France
bAssistance Publique-Hôpitaux de Paris, Groupe Hospitalier Pitié-Salpêtrière, Service de Pharmacie, 47-83 Boulevard de l'Hôpital, 75013 Paris, France
cAssistance 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
dUniversité Paris-Sud, UFR de Pharmacie, SAMM – Service d'Analyse des Médicaments et Métabolites, Institut d'Innovation Thérapeutique, 5, rue Jean Baptiste Clément, 92296 Châtenay-Malabry, France
eUniversité Paris-Descartes, UFR de Pharmacie, 4 Avenue de l'Observatoire, 75006 Paris, France. E-mail: bernard.do@parisdescartes.fr; Fax: +33 146691492; Tel: +33 662306275
First published on 9th April 2015
Apixaban is a novel anticoagulant drug acting as a direct, selective and reversible inhibitor of the coagulation factor Xa. Forced degradation under stress conditions were carried out in order to establish its stability profile. The drug was shown to be stable under photolytic, thermolytic and oxidative conditions, while under hydrolytic conditions, up to seven degradation products were generated for about 15% of drug degradation. The degradation products have been detected by linear gradient reversed phase high-performance liquid chromatography coupled with a photo diode array and with electrospray ionization tandem mass spectrometry. A combination of multistage mass spectrometry and high-resolution mass spectrometry (HR-MS) allowed the structural elucidation. The product ions of the degradation products were compared to those of the apixaban protonated ion so as to assign the most structures possible. This required a study in depth of the drug's fragmentation pattern, which has not been reported so far. In view of the products formed, it appears that hydrolysis of the oxopiperidine moiety of apixaban occurred in acidic medium, whereas that of the tetrahydro-oxo-pyridine moiety would further happen under alkaline conditions. Asides from characterization, the LC method was shown to indicate stability and validated as per the criteria described by the ICH guidelines.
LC-MS/MS methods were developed to determine apixaban alone in plasma or in the presence of its major metabolites7–11 to support clinical uses. Assay in tablets and simultaneous determination with other drugs using HPLC was also described.12,13 A literature survey, however, did not reveal any further information about the stability profile of apixaban or about its potential degradation products likely to form in time and/or under stress conditions. As drug may undergo degradations, leading potentially to activity loss or to occurrence of adverse effects associated with the appearance of degradation products, thorough knowledge of drug's stability profile is one of the key factors to prevent those risks during manufacturing, transportation and storage.
In this paper, we have focused on the identification and the characterization of degradation products generated in solution. Liquid chromatography combined with mass spectrometry has been well established and found to be a very useful technique for the identification and characterization of DPs.14–17 That's why high performance liquid chromatography coupled with multistage mass spectrometry (HPLC-MSn) was used. Different stress conditions were applied in order to simulate the degradation of active pharmaceutical substances, for which degradation can occur via many pathways such as basic and acidic hydrolysis, oxidation, photo-degradation or thermal degradation. The structures of observed degradation products were elucidated using multistage mass spectrometry and high-resolution mass spectrometry (HR-MS). A study in depth of apixaban fragmentation pattern was also achieved in order to help assign, by comparison, the structures of the major product ions coming from the degradation products ions. In addition, LC-UV method for quantitative determinations of apixaban in the presence of its degradation products has been validated as per ICH.18
Time (min) | A: water (% v/v) | B: acetonitrile (% v/v) |
---|---|---|
0 → 2 | 95 | 5 |
2 → 13 | 95 → 40 | 5 → 60 |
13 → 19 | 40 | 60 |
19→ 20 | 40 → 95 | 60 → 5 |
Detection and characterization were performed by mass spectrometry and high-resolution mass spectrometry. In both cases, an electro-spray ionisation (ESI) source operated in positive ion mode. In MS, the ionization conditions were set as following: ion spray-voltage was set at 5.5 kV, curtain gas (N2) flow rate at 40 psi, nebulizer gas (air) flow rate at 30 psi and heater gas (air) flow rate at 50 psi. Temperature was set at 500 °C. Nitrogen was used as collision and damping gas. Acquisition in full scan mode over the mass range of 50–550 Da was performed for the detection of the degradation products. MSn experiments for structural elucidation were carried out using 30% (arbitrary units) collision energy level (CEL). MS data were treated with Analyst® software version 1.5.2 and MS Manager® software version 12 (ACD Labs, Toronto, Canada). In HR-MS, the ionization conditions were set as follows: the source voltage was set at 3.4 kV and the temperatures were fixed at 53 °C (source) and 300 °C (capillary). S-Lens was set at 60%. Acquisition in full scan mode over the mass range of 300–550 Da was performed for the determination of the degradation products accurate masses. Data were treated with Xcalibur® software (version 2.2 SP 1.48).
A Q-SUN XE-1 Xenon test chamber (LX 5080 Q-Lab Westlake, California, USA) was used for photo-degradation studies.
Stress condition | Time | Average assay of API (% w/w, n = 3) | Average total impurities (% w/w, n = 3) | Average mass balance (assay + total impurities %, n = 3) | Commentaries |
---|---|---|---|---|---|
Acid hydrolysis (0.1 M HCl) | 24 hours | 84.9 | 14.6 | 99.5 | Degradation accompanied by appearance of DP-2, DP-3, DP-4, DP-5, DP-6 and DP-7 |
Base hydrolysis (0.1 M NaOH) | 3 hours | 88.1 | 10.8 | 98.9 | Degradation accompanied by appearance of DP-1 and DP-2 |
Oxidation (3% H2O2) | 72 hours | 99.3 | N.D. | 99.3 | No degradation occurred |
Thermal (80 °C) | 7 days | 99.5 | N.D. | 99.5 | No degradation occurred |
Photolysis (UV light) | 36 hours | 98.6 | N.D. | 98.6 | No degradation occurred |
As a result, the method could be used for the assay determination with implementation of system suitability testing criteria, i.e. Rs (apixaban/DP-4, ≥1.3), As (≤1.2) and N (≈4500).
Same chromatographic conditions were applied for the characterization of the degradation products by LC-ESI-HR-MSn (Table 1).
In total, seven degradation products were detected in the solutions subjected to hydrolysis, when taken at a degradation rate still inferior to 15%. Even if degradation continued beyond 15%, we have limited the present study to that of the degradation products formed precociously in the stress conditions, insofar as the others, sometimes secondarily formed, can be considered as less likely with respect to real-storage conditions.19 The studied degradation products are named “DPn”, where n accounts for the elution order. Base hydrolysis resulted DP-1 and DP-2 with relative retention times of 0.65 and 0.73, whereas acidic degradation chromatograms showed DP-2, DP-3, DP-4, DP-5, DP-6 and DP-7 with relative retention times of 0.73, 0.96, 1.01, 1.07, 1.16 and 1.21 (Fig. 1, Table 2). Aside from these DPs, the acidic degradation chromatogram also highlighted the presence of two other but much less intense compounds, eluted about 10.9 min. But unlike the other ones, they were not detected in mass spectrometry and therefore, cannot be studied or structurally elucidated.
Product ions m/z | Origin | Best possible elemental formula | Theorical masses m/z | Accurate masses m/z | Error (ppm) |
---|---|---|---|---|---|
460 | [M + H]+ | C25H26N5O4+ | 460.19793 | 460.19655 | −3.00 |
461 | MS2 (460 →) | C25H25N4O5+ | 461.18195 | 461.18054 | −3.06 |
443 | MS2 (460 →) | C25H23N4O4+ | 443.171382 | 443.17006 | −2.98 |
415 | MS3 (460 → 443 →) | C24H23N4O3+ | 415.176467 | 415.17547 | −2.40 |
387 | MS4 (460 → 443 → 415 →) | C23H23N4O2+ | 387.18155 | 387.18073 | −2.12 |
282 | MS3 (460 → 443 →) | C16H16N3O2+ | 282.123703 | 282.12279 | −3.24 |
264 | MS4 (460 → 443 → 282 →) | C16H14N3O+ | 264.11370 | 264.1122 | −5.68 |
254 | MS4 (460 → 443 → 282 →) | C15H16N3O+ | 254.128789 | 254.12791 | −3.46 |
241 | MS3 (460 → 443 →) | C13H9N2O3+ | 241.06077 | 241.06004 | −3.03 |
240 | MS4 (460 → 443 → 282 →) | C14H14N3O+ | 240.11314 | 240.11228 | −3.58 |
227 | MS4 (460 → 443 → 282 →) | C14H15N2O+ | 227.11789 | 227.11711 | −3.43 |
199 | MS3 (460 → 443 →) | C12H11N2O+ | 199.086589 | 199.08620 | −1.95 |
185 | MS4 (460 → 443 → 241 →) | C11H9N2O+ | 185.070939 | 185.07035 | −3.18 |
172 | MS4 (460 → 443 → 199 →) | C11H10NO+ | 172.07569 | 172.07516 | −3.08 |
Under positive ESI-MS conditions, [M + H]+ and [M + Na]+ ions were detected at m/z 460 and 482, respectively. Among the nitrogen functions apixaban is composed of, only carboxamide-amine function exhibits basic properties with a pKa of 13.1. It is therefore more amenable to protonation than are the others regarding the ionization process. The ESI-HRMS2 spectrum of (M + H)+ ion yielded 2 product ions with m/z of 461 and 443 (Table 3, Fig. 2a). The first ion would have been formed by hydrolysis affording a carboxylate derivative (C25H25N4O5+), while the second would be derived from deamination. Acylium derivative C25H23N4O4+ was also shown to have been produced from m/z 461 ion by loss of H2O when this one was taken as precursor during MS3 study (data not shown). Subjected to the MS3 process, C25H23N4O4+ (m/z 443) gave rise to the formation of four major product ions with m/z of 415, 282, 241 and 199 (Table 3, Fig. 2b). The other product ions were detected at a much lower intensity and as discussed later, they turn out to have originated from the previous ones (Fig. 3).
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Fig. 2 High-resolution MS2 spectrum of (a) the protonated ion of apixaban and (b) high-resolution MS3 spectrum of the product ion at m/z 443. |
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Fig. 3 High-resolution MS4 spectra of the product ions at (a) m/z 415, (b) m/z 282, (c) m/z 241 and (d) m/z 199. |
Transition 443 → 415 may be related to CO departure by heterolytic cleavage of 3C and the acyl carbon bond. Carbocation C24H23N4O3+ was formed. In turn, C24H23N4O3+ only generated one significantly intense MS4 product ion (C23H23N4O2+) at m/z 387, by loss of CO (Table 3, Fig. 3a). According to the scheme (Fig. 4), it was proposed that such a neutral loss would come from the oxo-piperidin moiety through a rearrangements cascade, triggered by migration of a hydrogen atom from 9C to 3C through 1,4 H-transfer. The conformation of C24H23N4O3+ is such that thereof would be quite stable by resonance.
Elimination of 2-(4-methoxyphenylimino)ethenone was proposed to explain the formation of m/z 282 ion (Fig. 4). The premise here was that another intermediate (2-(4-methoxyphenyl)-7-oxo-6-(4-(2-oxopiperidin-1-yl)phenyl)-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyridine-3-carbonyl ion) was formed through a rearrangement such as transamination, as illustrated in Fig. 4. From there, hydrogen migration from 8′C to the H-bond acceptor 1′N, through 1,6 H-transfer, would have led to the pyrazole ring opening by 1′N–2′N bond cleavage, to the formation of a π bond between 2′N and 3′C, and to the switch of the adjacent double bond. Eventually, the 161 Da moiety would have been released by heterolytic rupture of 4′C–3′C bond. When taken as precursor for MS4 study, C16H16N3O2+ (m/z 282) yielded the product ions at m/z 264, 254 and 240 (Table 1, Fig. 3b), likely by dehydration, CO elimination and by loss of H2CC
O, respectively. Furthermore, as stated in the proposed fragmentation scheme of apixaban (Fig. 4), the product ion at m/z 227 would have been generated from m/z 254 ion by expulsion of HCN.
As for the MS3 product ion detected at m/z 241 (Fig. 2b), it seemed to have been formed by loss of a 202 Da moiety, which could in all likelihood be attributed to 1-(4-(methyleneamino)phenyl)piperidin-2-one. As shown in Fig. 4, hydrogen atom migration from 8C to the H-bond acceptor 1N through 1,6 H-transfer, accompanied by a switch of 2N–3C single bond and of the adjacent double bonds, might have led in the first stage, to the opening of tetrahydropyridine ring. Next, a similar H-transfer process, that took place between 2′′N and electron-deficient 8′′C, would have ended up releasing the aforementioned neutral fragment by heterolytic cleavage of 8′′C–9′′C bond. Under the MS4 conditions (Fig. 3c), C25H23N4O4+ appeared to lose two CO to afford the product ion at m/z 185 (C11H9N2O+).
The last other important fragmentation route of C25H23N4O4+ (m/z 443) was represented by transition 443 → 199. Thereof would be formed by loss of a 216 Da moiety and of CO. 1-(4-Isocyanatophenyl)piperidin-2-one could possibly account for the so-called 216 Da moiety. Indeed, it was proposed that by tautomery, electron-deficient 5C had withdrawn a hydrogen atom to 9C, leading to the formation of a π bond between 4C and 9C. From there, switch of C9–C8 and 7N–8C single bonds would have allowed generating a metastable ion with m/z of 227, which in turn, would have undergone CO loss to afford m/z 199 ion (Fig. 4). When taken as precursor for MS4 study, m/z 199 ion could notably produce m/z 172 ion by elimination of HCN (Fig. 3d).
Throughout this study, the product ions presented in Fig. 4 were all confirmed by accurate mass measurement.
Products | rT (min) | Conditions | [M + H]+ | Elemental compositions | Theoretical masses | Accurate masses with error in ppm | Relevant MS/MS product ions assigned (m/z) |
---|---|---|---|---|---|---|---|
DP-1 | 8.7 | Basic | 478 | C25H28N5O5+ | 478.20849 | 478.20755 (−1.97) | 461; 460; 417; 288; 271; 244; 227; 203; 199; 191 |
DP-2 | 9.7 | Acidic/basic | 461 | C25H25N4O5+ | 461.18195 | 461.18090 (−2.28) | 443; 415; 387; 282; 254; 227; 199; 185; 172 |
DP-3 | 12.8 | Acidic | 379 | C20H18N4O4+ | 379.14008 | 379.13883 (−3.30) | 361; 333; 303; 241; 200; 199; 185; 172 |
Apixaban | 13.3 | — | 460 | C25H26N5O4+ | 460.19793 | 460.19674 (−2.59) | See Table 1 |
DP-4 | 13.6 | Acidic | 478 | C25H28N5O5+ | 478.20849 | 478.20702 (−3.07) | 461; 460; 443; 432; 416; 404; 390; 378; 361; 333; 300; 282; 199; 185; 101 |
DP-5 | 14.6 | Acidic | 393 | C21H21N4O4+ | 393.15573 | 393.15436 (−3.48) | 393; 379; 361; 333; 241; 200; 199; 185; 172; 156 |
DP-6 | 15.6 | Acidic | 475 | C26H27N4O5+ | 475.19760 | 475.19621 (−2.93) | 475; 461; 443; 282; 254; 241; 227; 199; 185; 172; 156 |
DP-7 | 16.2 | Acidic | 492 | C26H30N5O5+ | 492.22415 | 492.22257 (−3.21) | 475; 432; 416; 404; 390; 378; 377; 371; 333; 314; 282; 227; 199; 185; 115 |
MS/MS product ions of DP-2 and DP-6 were almost all the same as that of apixaban. Only the protonated precursor ions were different (Table 4). Instead of transition 460 → 443 corresponding to loss of NH3, transitions 461 → 443 and 475 → 443 took place for DP-2 and DP-6, respectively. They might correspond to dehydration and loss of methanol (Fig. 4). As a result, DP-2 and DP-6 might correspond to 1-(4-methoxyphenyl)-7-oxo-6-(4-(2-oxopiperidin-1-yl)phenyl)-4,5,6,7-tetrahydro-1H-pyrazolo[3,4-c]pyridine-3-carboxylic acid and to methyl-1-(4-methoxyphenyl)-7-oxo-6-(4-(2-oxopiperidin-1-yl)phenyl)-4,5,6,7-tetrahydro-1H-pyrazolo[3,4-c]pyridine-3-carboxylate, respectively. These structures were also confirmed by the accurate mass measure of DP-2 and DP-6 (Table 4).
As DP-1, DP-4 yielded a protonated ion with m/z of 478. Therefore, it could equally be considered as a hydrolysis product (Table 4). Its MS2 spectrum includes common ions with that of apixaban as was already mentioned for DP-1. It also displays extra product ions with m/z of 432, 416, 404, 390, 378, 361, 333, 300 and 101 (Fig. 5b). The presence of some of them confirmed that the oxo-piperidin ring opening had occurred by hydrolysis of the amide bond. The ion at m/z 101 may be due to N–C bond heterolytic cleavage releasing n-pentanoic acid carbocation. The product ion at m/z 378 would have been formed through N-dealkylation after the protonation of aniline–amine function. The anilinium derivative could in turn successively lose NH3 and CO to yield m/z 361 and m/z 333 ions, respectively. As shown in Fig. 7, existence of some of the product ions could be related to the C–C bonds rupture on the lateral chain. This seems to concern the product ions at m/z 432, 416 and 404. Aside from the fragmentation paths involving the lateral chain, elimination of 2-(4-methoxyphenylimino)ethenone that was already described for apixaban, would have generated m/z 300 ion, which in turn, would have lost a water molecule to afford m/z 282 ion. Similarly, loss of an isocyanatophenyl derivative along with CO would have formed m/z 199 ion. Therefore, such a fragmentation pattern is entirely consistent with the protonated ion of 5-(4-(3-carbamoyl-1-(4-methoxyphenyl)-7-oxo-4,5-dihydro-1H-pyrazolo[3,4-c]pyridin-6(7H)-yl)phenylamino)pentanoic acid, as precursor.
DP-7 protonated ion was detected at m/z 492. Its accurate mass, measured by HR-MS, is consistent with C30H26N5O5+ elemental formula (Table 4, Fig. 5c). Given a perfect parallelism between the fragmentation patterns of DP-4 and DP-7 (Table 2, Fig. 5 and 7), DP-7 was identified as methyl 5-(4-(3-carbamoyl-1-(4-methoxyphenyl)-7-oxo-4,5-dihydro-1H-pyrazolo[3,4-c]pyridin-6(7H)-yl)phenylamino)pentanoate.
DP-3 and DP-5 were also found to include the characteristic fragments m/z 361 and m/z 333 within their ESI-MS/MS spectra. Moreover, by comparing the elemental formula of protonated DP-3 and of DP-5 to that of the drug (Table 4), it was easy to demonstrate that they corresponded to aniline derivatives having lost the oxo-piperidin group. In addition, it seems that DP-3 would carry a carboxylate group and DP-5 a carboxymethyl group instead of the initial carboxamide function (Table 4). These assumptions were further supported by the determination of their fragmentation pattern such described by Fig. 8. Loss of water and of methanol from protonated DP-3 and DP-5, respectively, was observed through the presence of m/z 361 ion on both MS2 spectra. The protonated ion of DP-3 also corresponds to a product ion of the protonated ion of DP-5 after demethylation. As the degradation products structure had preserved the tetrahydropyridine–pyrazolo–methoxyphenyl core, it was logical that the transition involving loss of 2-(4-methoxyphenylimino)ethenone (361 → 200) was one more time detected. Always from the product ion at m/z 361, m/z 241 and m/z 199 ions would have been produced according to the same mechanisms as those described for apixaban, as shown in Fig. 8. As a result, DP-3 might correspond to 6-(4-aminophenyl)-1-(4-methoxyphenyl)-7-oxo-4,5,6,7-tetrahydro-1H-pyrazolo[3,4-c]pyridine-3-carboxylic acid and DP-5, to methyl-6-(4-aminophenyl)-1-(4-methoxyphenyl)-7-oxo-4,5,6,7-tetrahydro-1H-pyrazolo[3,4-c]pyridine-3-carboxylate.
A stability-indicating LC method was developed and it has shown suitable for the drug quantification as well as for the impurity determination.
Footnote |
† The first 2 authors contributed equally to this study and are therefore considered as first authors. |
This journal is © The Royal Society of Chemistry 2015 |