Journal of Pharmaceutical and Biomedical Analysis 94 (2014) 71–76
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Journal of Pharmaceutical and Biomedical Analysis journal homepage: www.elsevier.com/locate/jpba
Identification, characterization, synthesis and HPLC quantification of new process-related impurities and degradation products in retigabine ˇ Michal Douˇsa a,∗ , Jan Srbek a , Stanislav Rádl a , Josef Cern y´ a , Ondˇrej Klecán a , Jaroslav Havlíˇcek a , Marcela Tkadlecová a , Tomáˇs Pekárek a , Petr Gibala a , Lucie Nováková b a b
Zentiva, k.s. Praha, U Kabelovny 130, 102 37 Praha 10, Czech Republic Department of Analytical Chemistry, Charles University, Faculty of Pharmacy, Heyrovského 1203, 500 05 Hradec Králové, Czech Republic
a r t i c l e
i n f o
Article history: Received 19 December 2013 Received in revised form 23 January 2014 Accepted 26 January 2014 Available online 3 February 2014 Keywords: Retigabine Process-related impurities Identification Synthesis HPLC
a b s t r a c t Two new impurities were described and determined using gradient HPLC method with UV detection in retigabine (RET). Using LC–HRMS, NMR and IR analysis the impurities were identified as RET-dimer I: diethyl {4,4 -diamino-6,6 -bis[(4-fluorobenzyl)amino]biphenyl-3,3 -diyl}biscarbamate and RET-dimer II: ethyl {2-amino-5-[{2-amino-4-[(4-fluorobenzyl) amino] phenyl} (ethoxycarbonyl) amino]-4-[(4fluorobenzyl)amino] phenyl}carbamate. Reference standards of these impurities were synthesized followed by semipreparative HPLC purification. The mechanism of the formation of these impurities is also discussed. An HPLC method was optimized in order to separate, selectively detect and quantify all process-related impurities and degradation products of RET. The presented method, which was validated in terms of linearity, limit of detection (LOD), limit of quantification (LOQ) and selectivity is very quick (less than 11 min including re-equilibration time) and therefore highly suitable for routine analysis of RET related substances as well as stability studies. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Retigabine (RET), which is N-(2-amino-4-(4fluorobenzylamino)phenyl) carbamic acid ethyl ester, is an antiepileptic drug with novel mechanism of action that involves opening of neuronal KV7.2–7.5 (formerly KCNQ2–5) voltageactivated K+ channels [1]. In vitro studies hinted that ezogabine might also exert its therapeutic effect through the augmentation of currents mediated by ␥-aminobutyric acid [2,3] moreover, it exhibits efficacy in a range of animal epilepsy models [4]. LC-UV [5] and LC–MS/MS [6–8] methods were developed and reported for the determination of RET and its N-acetyl or N-glucuronide metabolites in biological samples. Four processrelated impurities: 2,4-diaminophenylcarbamate (Imp 1), ethyl 2-amino-4-(benzylamino)phenylcarbamate (Imp 2), ethyl 2(Imp-3), acetamido-4-(4-fluorobenzylamino)phenylcarbamate ethyl 4-(4-fluorobenzylamino)-2-nitrophenylcarbamate (Imp4) were identified by liquid chromatography–tandem mass
∗ Corresponding author. Tel.: +420 720520506. E-mail address: [email protected] (M. Douˇsa). 0731-7085/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jpba.2014.01.042
spectrometry using electrospray ionization and quadrupole time-of-flight mass analyzer (LC–ESI-Q-TOF-MS) [9]. This paper describes identification, synthesis and determination of the two new impurities in RET which were found in the drug substance using gradient HPLC method with UV detection at 247 nm. The proposed chemical structures of these impurities were confirmed by synthesis, followed by characterization using MS, NMR and IR analysis. The formation of these impurities was proposed with the respect to the knowledge of chemical synthesis. 2. Experimental 2.1. Reagents and chemicals Acetonitrile HPLC gradient grade (J.T. Baker, USA) and water purified by Milli-Q system (Millipore, USA) were used for preparation of samples, reference solutions and mobile phases. All other chemicals were of analytical grade or pure grade quality (Sigma–Aldrich, Czech Republic). Three different batches of RET were prepared in Zentiva k.s. (Czech Republic). The reference standards of impurities, 2,4-diaminophenylcarbamate (Imp 1), ethyl 2-amino-4-(benzylamino)phenylcarbamate (Imp 2) were obtained from Matrix Laboratories Limited (India).
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2.2. Preparation of standard and sample solutions The standard of RET (in house standard, purity 99.8%) was dissolved in acetonitrile to obtain standard stock solution (1.5 mg/ml). The system suitability test solution was prepared by spiking RET standard stock solution with Imp 2 to obtain concentration of 0.15% with respect to RET. The reference solution was prepared by diluting RET standard stock solution to obtain concentration of 2.25 g/ml (concentration of 0.15% with respect to RET sample solution). Preparation of RET-dimer I and RET-dimer II impurities is described in Section 3. 15 mg of RET sample was weighed into a 10 ml volumetric flask and 8 ml of acetonitrile was added. The sample was dissolved and sonicated for 2 min in an ultrasonic bath UCC4 (TESON, Slovakia) to obtain RET preparation solution. After cooling to the laboratory temperature, the flask was filled to the mark by acetonitrile. 2.3. HPLC instrumentation and methods All chromatographic experiments were carried out on an Acquity UPLC system with a photodiode array detector (Waters, USA). The system control, data acquisition and processing was accomplished by the Empower software (Waters, USA). Chromatographic separations were performed on Ascentis Express C18 column (100 mm × 4.6 mm, 2.7 m; Sigma, Czech Republic). The gradient elution employed solutions A and B as mobile phase components. The solvent A was 10 mM potassium hydrogenphosphate, pH was adjusted to 7.5 using 5 M phosphoric acid, while solvent B was acetonitrile. The mobile phase was pumped at 1.2 ml/min and the column was thermostated at a temperature of 40 ◦ C. The gradient programme was set as follows: time/% of solvent B: 0/25, 0.5/25, 7.5/77, 9.5/77, 10/25 with an equilibration time of 1 min. The injection volume was 1 l and analytes were monitored at a wavelength of 247 nm. 2.4. LC–MS instrumentation and methods High-resolution MS (HRMS) experiments were performed on a LTQ Orbitrap Mass Spectrometer (Thermo, San Jose, USA) coupled to an HPLC HTS PAL system (CTC Analytics, Switzerland). LC separation was performed on a Kinetex C18, 150 mm × 4.6 mm, 2.6 m (Phenomenex, Torrance, USA) column using 0.6 ml/min flow rate and mobile phase consisting of 10 mM ammonium formate (pH 6.3) and acetonitrile (gradient of acetonitrile ranging from 30% to 100% in 18 min). For the ionization of the analytes an ESI ion source was operated in the positive ion mode (desolvation temperature 400 ◦ C, capillary temperature 300 ◦ C, discharge current 4 A and tube lens voltage 40 V). 2.5. Semipreparative instrumentation and methods Preparative HPLC separation and fraction collection was carried out on a Waters Autopurification system (System fluidics organizer, 2545 binary gradient module, 2767 sample manager, 515 HPLC pump, MS and 2487 dual-wavelength absorbance detector; Waters, USA). The flow rate of 20 ml/min was employed throughout the preparation. The injection volume was 500 l. In order to monitor UV signal from the semipreparative column, the effluent was splitted in the ratio 1:1000 into the methanol flow from 515 HPLC pump, which was directed to MS and UV detector. The fraction collection was triggered by setting a minimal intensity threshold (MIT = 500,00 V for UV detection, MIT = 1,000,000 for MS detection) of the UV signal at 247 nm. A semipreparative XBridge Prep C18 OBD column (100 mm × 19 mm, 5 m; Waters, USA) was used for preparative purposes using mobile phase consisting of 0.025% aqueous solution of ammonium hydroxide (solvent A) and
Fig. 1. The chromatogram of RET with relevant impurities under method conditions on an Ascentis Express C18 column (100 mm × 4.6 mm, 2.7 m; Sigma, Czech Republic).
acetonitrile (solvent B). The separation was employed using gradient elution based on following programme: time/% of solvent B: 0/40, 0.5/40, 5.25/75, 5.35/100, 6.55/10, 6.95/40 with an equilibration time of 0.55 min. 2.6. NMR, IR and other instrumentation and methods Nuclear magnetic resonance (NMR) spectra were obtained using a Bruker Avance 500 (Bruker Biospin, Germany) at 500.13 MHz (1H) and 125.78 MHz. All NMR experiments were performed in dimethyl sulfoxide-d6 or in acetonitrile-d6 at 298 and 343 K. At 500 MHz, standard 5 mm TBI (triple-broadband inverse) probehead equipped with z-gradient coils was employed for all measurements. IR spectra were measured with the single-reflection ATR (ZnSe) FTIR spectrometer Nicolet Nexus (Thermo, USA). The spectra were acquired by accumulation of 64 scans with 2 cm−1 resolution. 3. Results and discussion 3.1. Identification of unknown impurities The unknown impurities with relative retention time (RRT) 1.38 and 1.50 with respect to RET were obtained in several batches of RET using gradient HPLC method with UV detection at 247 nm (Fig. 1). LC–MS analysis detected ions corresponding to the two compounds, including protonated molecular ion [M+H]+ at m/z = 605.2681 (compound with RRT 1.38) and 605.2680 (compound with RRT 1.50). Use of the HRMS in this measurement allowed determination of the corresponding elemental composition (C32 H34 N6 O4 F2 ) of the both unknown compounds. Moreover, full scan MS spectra and corresponding MS/MS spectra of each compound contained the same molecular ion but different fragments. These findings in the fragmentation pattern hinted the different structure of the corresponding isobaric compounds. Full scan MS spectrum and corresponding MS/MS spectra (at low collision energies) of the compound with RRT 1.38 contained only one dominant signal of the molecular ion, which corresponded to the relatively strong bond between two aromatic rings in the molecule (Fig. 2A). On the other hand, spectra of the compound with RRT 1.50 (recorded at the same experimental conditions as for compound with RRT 1.38) contained two major fragments m/z = 389.1621 (C19 H22 N4 O4 F) and m/z = 302.1301 (C16 H17 N3 O2 F), which can be explained by the connection of the RET monomers through the nitrogen atom (Fig. 2B). The structures of these impurities were verified by NMR analysis after semipreparative isolation.
M. Douˇsa et al. / Journal of Pharmaceutical and Biomedical Analysis 94 (2014) 71–76
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Fig. 2. Positive ion mode ESI full scan mass spectra of RET-dimer I (A) and RET-dimer II (B).
The proposed structure of the compound with RRT 1.38 was diethyl {4,4 -diamino-6,6 -bis[(4-fluorobenzyl)amino]biphenyl3,3 -diyl}biscarbamate (RET-dimer I) and the propose structure of the compound with RRT 1.50 was ethyl {2-amino-5-[{2amino-4-[(4-fluorobenzyl)amino]phenyl}(ethoxycarbonyl) amino]-4-[(4-fluorobenzyl)amino]phenyl} carbamate (RET-dimer II). The assignment of NMR signals was performed for both dimeric impurities by means of 1 H, 13 C and 2D (COSY, HSQC and HMBC) spectra. The given structures were unambiguously confirmed (Table 1). If the NMR spectra of the symmetric dimer were confirmed with those of original RET one it was clear that the fluorinated aromatic ring remained untouched and that the two remaining protons (H3 and H6) at the other aromatic ring were in para position to each other. Similarly for nonsymmetric dimer it could be confirmed that the new bond was formed between C4 of one retigabine and N8 of the other. Although the pure RET is a colourless compound, the crude product was intensively coloured. Similarly, RET changed quickly colour from red to dark violet when left on air. This suggests that some quinone diimine structures (supplementary material; supplemental 1) could be involved. Formation of dimers of various substituted 1,4-diaminobenzenes via the corresponding quinone diimines is well demonstrated. The best example is the oxidative selfcoupling of 1,4-diaminobenzene leading to the Bandrowski’s base [10] or reaction of 1,4-diaminobenzene with 1,3-diaminobenzene derivatives under oxidative conditions (supplementary material; supplement 2), which is used also in oxidative hair dye formation
[11]. Since the last steps of the synthesis were done under reductive conditions (supplementary material; supplement 3), these reactions leading to the dimeric product formation could probably take place during work-up and following manipulations. Primarily formed oxidative intermediate could then provide both dimeric impurities. Formation of RET-dimer II, where a C-N bond was formed, was very similar to reactions, which were described in Ref. [10] (supplementary material; supplement 2). On the other hand, formation of RET-dimer I, where a C-C bond was formed, assumed nucleophilic addition of a carbon nucleophile to an activated quinone diimine structure (Fig. 3). Though this type of reaction was not widely reported, some examples were published [12]. Formation of similar dimeric metabolites of flupirtine by similar mechanism involving the quinone diimines has been reported as well [13]. 3.2. Synthesis and characterization of reference standards of impurities 3.2.1. Preparation of RET-dimer I and RET-dimer II Although reductive conditions were used in the final steps of the RET synthesis, the proposed structures of the RET dimeric impurities suggested rather oxidative conditions for their formation. The procedure of synthesis of dimeric impurities was following: A solution of potassium persulfate (K2 S2 O8 , 2.9 g, 10.7 mmol) in 50% aqueous acetonitrile (200 ml) was added during 30 min to a stirred solution of RET (10.0 g, 33.0 mmol) in 50% aqueous acetonitrile (200 ml). The resulting dark purple reaction mixture was stirred
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Table 1 NMR assignments for RET dimers. Position
␦(C)
␦(H)
RET-dimer I 1 2 3 4 5 6 7 8 9 11 12 13 14 = 18 15 = 17 16 20 21
144.9 114.1 131.3 114.7 146.5 99.3 (br.) – – 156.7 – 47.5 137.4 130 116 162.8 61.7 15.1
– – 6.65 – – 5.98 4.04 6.7 – 4.42 4.22 – 7.29 7.02 – 4.08 1.22
RET-dimer II 1 1 2 2 3 3 4 4 5 5 6 6 7; 7 8 9; 9 11; 11 12 12 13; 13 14 = 18; 14 = 18 15 = 17; 15 = 17 16; 16 20; 20 21; 21
143.5 141.6 119.3 118.7 127.7 125.7 103 112.4 147.5 141.6 99.9 97.9 – – 154.5; 154.6 – 45.3 45.7 135.2; 136.6 128.3; 128.5 114.3; 114.4 160.7; 160.8 59.4; 60.7 14.05; 14.10
– – – – 6.76 6.93 5.96 – – – 6.05 5.92 4.45 7.91 – 5.75 4.19 4.21 – 7.18; 7.38 6.99; 7.08 – 4.06; 4.08 1.13; 1.21
Mult.
Integ. H
s
2
s s s (broad)
2 4 2
t d
2 4
m m
4 4
quartet t (broad)
4 6
∼7 ∼7
d s dd
1 1 1
8.5
d s s (broad) s (broad)
1 1 4 1
2.5
t (broad) d d
1; 1 2 2
m; m m; m
2; 2 2; 2
q; q t; t
2; 2 3; 3
Note: RET-dimer I: ␦ in ppm, J in Hz. Reference for 1 H: acetonitrile-d6 (1.94 ppm), for dimethyl sulfoxide-d6 (2.50 ppm), for 13 C: DMSO-d6 (39.50 ppm).
13
J(H.H)
J(C.F)
6.2 6.2 3 8 21 244
8.6; 2.5
6.2 6 3.0; 3.0 8.0; 8.0 21.1; 21.1 242.0; 242.0 7.0; 7.0 7.1; 7.1
C: acetonitrile-d6 (1.39 ppm); RET-dimer II: ␦ in ppm, J in Hz. Reference for 1H:
for further 10 min, then diluted with water (1000 ml) and extracted with ethyl acetate (3× 500 ml). The combined extracts were washed with water (500 ml), dried over magnesium sulfate and evaporated under reduced pressure. The obtained residue (8.6 g) contained 16.1% of RET-dimer I and 15.7% of RET-dimer II. The product was dissolved in acetonitrile to obtain concentration of 100 mg/ml and subsequently purified by semipreparative HPLC. The collection parameters of the semipreparative HPLC method were optimized with respect to high concentration of RET-dimers in the solution of crude sample. The solution of crude sample was injected into the semipreparative column and the fractions of dimeric impurities were repeatedly collected and combined. The combined fractions were evaporated under vacuum at 50 ◦ C to dryness to obtain pure solid dimeric impurity standards.
RET-dimer I of IR spectra were confirmed by following characteristic absorption maxima: (N H) 3470, 3379, 3362, 3317, (C O) 1724, 1699, (C C) 1626, 1443, ␦(N H) 1527, 1505, (C O) + (C F) 1219, (C O) 1149 cm−1 , the main functional groups of RET-dimer II of IR spectra were confirmed by following characteristic absorption maxima: (N H) 3344, (C O) 1686, (C C) 1602, ␦(N H) 1507, (C O) + (C F) 1217, (C O) 1155 cm−1 . Finally, all impurity reference standards of impurities were quantitatively characterized. The potency of the standards was calculated based on the values obtained from determination of impurities (organic, inorganic, water and residual solvents) by applying the principle of mass balance. The potency of RET-dimer I impurity was 99.2% and potency of RET-dimer II impurity was 91.6%.
3.2.2. The characterization reference standards of impurities The structures of prepared dimeric impurities were confirmed by MS and NMR analysis under the conditions described in Section 3.1 and obtained data were in agreement with the data measured for impurities isolated from RET sample. The qualitative characterization of reference standards was also performed using IR measurements (Fig. 4). The main functional groups of
3.3. Optimization of HPLC-UV method The composition and gradient profile of the mobile phase was optimized to achieve the retention factor of RET k ≥ 3.0, symmetry factor AS ≤ 1.5 and resolution between Imp 2 and RET RS ≥ 2.5. The dead volume of column used was determined using injection of sodium nitrate. The chromatographic data were calculated in
M. Douˇsa et al. / Journal of Pharmaceutical and Biomedical Analysis 94 (2014) 71–76
75
Fig. 3. Proposed mechanism of formation of dimeric impurities of RET.
agreement with European Pharmacopoeia (Ph.Eur.) [14]. Acetonitrile was selected as sample solvent. The separation of RET and its dimeric impurities was optimized by varying acetonitrile content, concentration and pH of potassium hydrogenphosphate buffer in the mobile phase. All experiments were performed using 10 mM phosphate buffer. The pH was varied in the range from 6.0 to 8.0. Acceptable resolution of tested impurities was achieved with gradient elution of acetonitrile as the organic modifier using flow rate 1.2 ml/min at temperature of 40 ◦ C and 10 mM buffer at pH of 7.5. The chromatogram of separation of RET and its dimeric impurities measured at the optimized HPLC conditions is shown in Fig. 1.
3.4. Validation of HPLC-UV method The method was validated according to ICH Q2(R1) guideline [15]. 3.4.1. System suitability The system suitability test was performed before each run to assure that the analytical method could be used with a satisfactory performance. Repeatability of injections expressed as relative standard deviation (RSD %) of peak area of RET and resolution between Imp 2 and RET for the five consecutive injections of a system
Fig. 4. IR spectra of RET-dimer I (A) and RET-dimer II (B).
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Table 2 The validation parameters of method. Parameter Linearity Slope (V/%) Intercept (V) Correlation coefficient QC coefficient LOD (%) LOQ (%)
4. Conclusion
RET
RET-dimer I
RET-dimer II
289 967 −10.9 0.999 2.0 0.001 0.004
384 404 −37.1 0.999 2.1 0.001 0.003
332 941 86.8 0.999 2.3 0.001 0.003
suitability test solution were limited to ≤1.0% and RS ≥ 2.5 respectively. For all the measurements performed during the validation, the resolution more than 3.0 and RSD of peak areas ≤0.4% was achieved. 3.4.2. Limit of detection (LOD), limit of quantification (LOQ) and linearity LOD and LOQ were calculated for all impurities based on signalto-noise ratio (S/N). The baseline noise was measured in a blank injection in the region of retention time of relevant impurity using chromatographic software. Calculated LOD and LOQ are listed in Table 1. A set of all six impurities and RET solutions at the concentration range from LOQ to 200% of the general specification limit (LOQ – 3.0 g/ml) were prepared. The calibration curves were constructed by plotting the peak area of a given analyte against its concentration. The calibration equations were calculated using linear regression analysis. The parameters of calibration curves are shown in Table 2. The linearity of the calibration curves was investigated using other statistical approaches such as the quality coefficient QC [16]. If the quality coefficient QC fulfilled the criterion QC < 5%, the linearity of calibration model was demonstrated (Table 1). The calculated parameters of calibration curves indicated satisfactory linearity. The correction response factor (CRF) of RET-dimer I and RET-dimer II with respect to RET at 247 nm was obtained from the slope ratio of the appropriate calibration curves. CRF for RET-dimer I was 0.75 and RET-dimer II was 0.83. 3.4.3. Method selectivity No interfering coeluting peaks in blank solvent were observed which demonstrated an adequate selectivity of HPLC method. The selectivity of this method was further evaluated using forced degradation studies. The samples were subjected to hydrolysis (water at 90 ◦ C for 8 h). The PDA detector was used to demonstrate the spectral peak purity of all degradation products. The formation of dimeric impurities, 4-fluorobenzaldehyde, impurity with RRT 0.68 and RRT 0.85 was observed. LC–HRMS high resolution measurement revealed ions corresponding to the unknown impurity with RRT 0.68, including protonated molecular ion [M+H]+ at m/z = 258.1040. Use of the high resolution MS in this measurement allowed determination of the corresponding elemental composition (C14 H12 N3 OF) of the unknown compounds. The proposed structure of this impurity was 5-[(4-fluorobenzyl)amino]-1,3dihydro-2H-benzimidazol-2-one. This impurity was found after the stress test only and was not observed in any of the RET samples. Therefore, this impurity was not characterized anymore. The proposed HPLC method was selective for the determination of process-related impurities in RET.
The new related compounds of RET were found using highly selective HPLC-UV method and identified by means of LC–MS, NMR and IR studies. These impurities were generated in the last step of the RET synthesis and were identified as diethyl {4,4 -diamino-6,6 -bis[(4-fluorobenzyl)amino]biphenyl3,3 -diyl}biscarbamate (RET-dimer I) and ethyl {2-amino-5[{2-amino-4-[(4-fluorobenzyl) amino] phenyl} (ethoxycarbonyl) amino]-4-[(4-fluorobenzyl)amino] phenyl}carbamate (RET-dimer II). The analytical standards of both RET-dimers were synthesized and fully characterized. The quick and efficient HPLC method for the measurement of RET and related-process impurities has been proposed. The method has various advantages over those previously reported, such as very short analysis time and high separation efficiency. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jpba.2014.01.042. References [1] G. Blackburn-Munro, W. Dalby-Brown, N.R. Mirza, J.D. Mikkelsen, R.E. Blackburn-Munro, Retigabine: chemical synthesis to clinical application, CNS Drug Rev. 11 (2005) 1–20. [2] M. Bialer, H.S. White, Key factors in the discovery and development of new antiepileptic drugs, Nature Rev. Drug Discov. 9 (2010) 68–82. [3] C. Rundfeldt, The new anticonvulsant retigabine (D-23129) acts as an opener of K+ channels in neuronal cells, Eur. J. Pharmacol. 336 (1997) 243–249. [4] A. Rostock, C. Tober, C. Rundfeldt, R. Bartsch, J. Engel, E.E. Polymeropoulos, B. Kutscher, W. Löscher, D. Hönack, H.S. White, H.H. Wolf, D-23129: a new anticonvulsant with a broad spectrum activity in animal models of epileptic seizures, Epilepsy Res. 23 (1996) 211–223. [5] A. Hiller, N. Nguyen, C.P. Strassburg, Q. Li, H. Jainta, B. Pechstein, P. Ruus, J. Engel, R.H. Tukey, T. Kronbach, Retigabine N-glucuronidation and its potential role in enterohepatic circulation, Drug Metab. Dispos. 27 (1999) 605–612. [6] G.M. Ferron, J. Paul, R. Fruncillo, L. Richards, N. Knebel, J. Getsy, S. Troy, Multiple-dose linear, dose-proportional pharmacokinetics of retigabine in healthy volunteers, J. Clin. Pharmacol. 42 (2002) 175–182. [7] W. Bu, M. Nguyen, C. Xu, C.C. Lin, L.T. Yeh, V. Borges, Determination of N-acetyl retigabine in dog plasma by LC/MS/MS following off-line microelution 96-well solid phase extraction, J. Chromatogr. B 852 (2007) 465–472. [8] N.G. Knebel, S. Grieb, S. Leisenheimer, M. Locher, Determination of retigabine and its acetyl metabolite in biological matrices by on-line solid-phase extraction (column switching) liquid chromatography with tandem mass spectrometry, J. Chromatogr. B 748 (2000) 97–111. [9] X. Wang, H. Zhou, J. Zheng, C. Huang, W. Liu, L. Yu, S. Zeng, Identification and characterization of four process-related impurities in retigabine, J. Pharm. Biomed. Anal. 71 (2012) 148–151. [10] E.A. Shilova, A. Heynderickx, O. Siri, Bandrowski’s base revisited: toward an unprecedented class of quinonediimines or new two-way chromophoric molecular switches, J. Org. Chem. 75 (2010) 1855–1861. [11] Scientific Committee on Consumer Safety SCCP/0941/05, in: Opinion on Exposure to Reactants and Reaction Products of Oxidative Hair Dye Formulations, 2005. [12] D.L. Boger, H. Zarrinmayeh, Regiocontrolled nucleophilic addition to selectively activated p-quinone diimines: alternative preparation of a key intermediate employed in the preparation of the CC-1065 left-hand subunit, J. Org. Chem. 55 (1990) 1379–1390. [13] K. Methling, P. Reszka, M. Lalk, O. Vrana, E. Scheuch, W. Siegmund, B. Terhaag, P.J. Bednarski, Drug Metab. Disp. 37 (2009) 479–493. [14] European Pharmacopoeia, Strasbourg: Council of Europe, 6th ed., 2009, pp. 4407–4413. [15] ICH Q2(R1), Validation of Analytical Procedures; Step 4 Version, 2005. [16] J. Van Loco, M. Elskens, Ch. Croux, H. Beernaert, Linearity of calibration curves: use and misuse of the correlation coefficient, Accredit. Qual. Assur. 7 (2002) 281–285.
Contents lists available at ScienceDirect
Journal of Pharmaceutical and Biomedical Analysis journal homepage: www.elsevier.com/locate/jpba
Identification, characterization, synthesis and HPLC quantification of new process-related impurities and degradation products in retigabine ˇ Michal Douˇsa a,∗ , Jan Srbek a , Stanislav Rádl a , Josef Cern y´ a , Ondˇrej Klecán a , Jaroslav Havlíˇcek a , Marcela Tkadlecová a , Tomáˇs Pekárek a , Petr Gibala a , Lucie Nováková b a b
Zentiva, k.s. Praha, U Kabelovny 130, 102 37 Praha 10, Czech Republic Department of Analytical Chemistry, Charles University, Faculty of Pharmacy, Heyrovského 1203, 500 05 Hradec Králové, Czech Republic
a r t i c l e
i n f o
Article history: Received 19 December 2013 Received in revised form 23 January 2014 Accepted 26 January 2014 Available online 3 February 2014 Keywords: Retigabine Process-related impurities Identification Synthesis HPLC
a b s t r a c t Two new impurities were described and determined using gradient HPLC method with UV detection in retigabine (RET). Using LC–HRMS, NMR and IR analysis the impurities were identified as RET-dimer I: diethyl {4,4 -diamino-6,6 -bis[(4-fluorobenzyl)amino]biphenyl-3,3 -diyl}biscarbamate and RET-dimer II: ethyl {2-amino-5-[{2-amino-4-[(4-fluorobenzyl) amino] phenyl} (ethoxycarbonyl) amino]-4-[(4fluorobenzyl)amino] phenyl}carbamate. Reference standards of these impurities were synthesized followed by semipreparative HPLC purification. The mechanism of the formation of these impurities is also discussed. An HPLC method was optimized in order to separate, selectively detect and quantify all process-related impurities and degradation products of RET. The presented method, which was validated in terms of linearity, limit of detection (LOD), limit of quantification (LOQ) and selectivity is very quick (less than 11 min including re-equilibration time) and therefore highly suitable for routine analysis of RET related substances as well as stability studies. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Retigabine (RET), which is N-(2-amino-4-(4fluorobenzylamino)phenyl) carbamic acid ethyl ester, is an antiepileptic drug with novel mechanism of action that involves opening of neuronal KV7.2–7.5 (formerly KCNQ2–5) voltageactivated K+ channels [1]. In vitro studies hinted that ezogabine might also exert its therapeutic effect through the augmentation of currents mediated by ␥-aminobutyric acid [2,3] moreover, it exhibits efficacy in a range of animal epilepsy models [4]. LC-UV [5] and LC–MS/MS [6–8] methods were developed and reported for the determination of RET and its N-acetyl or N-glucuronide metabolites in biological samples. Four processrelated impurities: 2,4-diaminophenylcarbamate (Imp 1), ethyl 2-amino-4-(benzylamino)phenylcarbamate (Imp 2), ethyl 2(Imp-3), acetamido-4-(4-fluorobenzylamino)phenylcarbamate ethyl 4-(4-fluorobenzylamino)-2-nitrophenylcarbamate (Imp4) were identified by liquid chromatography–tandem mass
∗ Corresponding author. Tel.: +420 720520506. E-mail address: [email protected] (M. Douˇsa). 0731-7085/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jpba.2014.01.042
spectrometry using electrospray ionization and quadrupole time-of-flight mass analyzer (LC–ESI-Q-TOF-MS) [9]. This paper describes identification, synthesis and determination of the two new impurities in RET which were found in the drug substance using gradient HPLC method with UV detection at 247 nm. The proposed chemical structures of these impurities were confirmed by synthesis, followed by characterization using MS, NMR and IR analysis. The formation of these impurities was proposed with the respect to the knowledge of chemical synthesis. 2. Experimental 2.1. Reagents and chemicals Acetonitrile HPLC gradient grade (J.T. Baker, USA) and water purified by Milli-Q system (Millipore, USA) were used for preparation of samples, reference solutions and mobile phases. All other chemicals were of analytical grade or pure grade quality (Sigma–Aldrich, Czech Republic). Three different batches of RET were prepared in Zentiva k.s. (Czech Republic). The reference standards of impurities, 2,4-diaminophenylcarbamate (Imp 1), ethyl 2-amino-4-(benzylamino)phenylcarbamate (Imp 2) were obtained from Matrix Laboratories Limited (India).
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2.2. Preparation of standard and sample solutions The standard of RET (in house standard, purity 99.8%) was dissolved in acetonitrile to obtain standard stock solution (1.5 mg/ml). The system suitability test solution was prepared by spiking RET standard stock solution with Imp 2 to obtain concentration of 0.15% with respect to RET. The reference solution was prepared by diluting RET standard stock solution to obtain concentration of 2.25 g/ml (concentration of 0.15% with respect to RET sample solution). Preparation of RET-dimer I and RET-dimer II impurities is described in Section 3. 15 mg of RET sample was weighed into a 10 ml volumetric flask and 8 ml of acetonitrile was added. The sample was dissolved and sonicated for 2 min in an ultrasonic bath UCC4 (TESON, Slovakia) to obtain RET preparation solution. After cooling to the laboratory temperature, the flask was filled to the mark by acetonitrile. 2.3. HPLC instrumentation and methods All chromatographic experiments were carried out on an Acquity UPLC system with a photodiode array detector (Waters, USA). The system control, data acquisition and processing was accomplished by the Empower software (Waters, USA). Chromatographic separations were performed on Ascentis Express C18 column (100 mm × 4.6 mm, 2.7 m; Sigma, Czech Republic). The gradient elution employed solutions A and B as mobile phase components. The solvent A was 10 mM potassium hydrogenphosphate, pH was adjusted to 7.5 using 5 M phosphoric acid, while solvent B was acetonitrile. The mobile phase was pumped at 1.2 ml/min and the column was thermostated at a temperature of 40 ◦ C. The gradient programme was set as follows: time/% of solvent B: 0/25, 0.5/25, 7.5/77, 9.5/77, 10/25 with an equilibration time of 1 min. The injection volume was 1 l and analytes were monitored at a wavelength of 247 nm. 2.4. LC–MS instrumentation and methods High-resolution MS (HRMS) experiments were performed on a LTQ Orbitrap Mass Spectrometer (Thermo, San Jose, USA) coupled to an HPLC HTS PAL system (CTC Analytics, Switzerland). LC separation was performed on a Kinetex C18, 150 mm × 4.6 mm, 2.6 m (Phenomenex, Torrance, USA) column using 0.6 ml/min flow rate and mobile phase consisting of 10 mM ammonium formate (pH 6.3) and acetonitrile (gradient of acetonitrile ranging from 30% to 100% in 18 min). For the ionization of the analytes an ESI ion source was operated in the positive ion mode (desolvation temperature 400 ◦ C, capillary temperature 300 ◦ C, discharge current 4 A and tube lens voltage 40 V). 2.5. Semipreparative instrumentation and methods Preparative HPLC separation and fraction collection was carried out on a Waters Autopurification system (System fluidics organizer, 2545 binary gradient module, 2767 sample manager, 515 HPLC pump, MS and 2487 dual-wavelength absorbance detector; Waters, USA). The flow rate of 20 ml/min was employed throughout the preparation. The injection volume was 500 l. In order to monitor UV signal from the semipreparative column, the effluent was splitted in the ratio 1:1000 into the methanol flow from 515 HPLC pump, which was directed to MS and UV detector. The fraction collection was triggered by setting a minimal intensity threshold (MIT = 500,00 V for UV detection, MIT = 1,000,000 for MS detection) of the UV signal at 247 nm. A semipreparative XBridge Prep C18 OBD column (100 mm × 19 mm, 5 m; Waters, USA) was used for preparative purposes using mobile phase consisting of 0.025% aqueous solution of ammonium hydroxide (solvent A) and
Fig. 1. The chromatogram of RET with relevant impurities under method conditions on an Ascentis Express C18 column (100 mm × 4.6 mm, 2.7 m; Sigma, Czech Republic).
acetonitrile (solvent B). The separation was employed using gradient elution based on following programme: time/% of solvent B: 0/40, 0.5/40, 5.25/75, 5.35/100, 6.55/10, 6.95/40 with an equilibration time of 0.55 min. 2.6. NMR, IR and other instrumentation and methods Nuclear magnetic resonance (NMR) spectra were obtained using a Bruker Avance 500 (Bruker Biospin, Germany) at 500.13 MHz (1H) and 125.78 MHz. All NMR experiments were performed in dimethyl sulfoxide-d6 or in acetonitrile-d6 at 298 and 343 K. At 500 MHz, standard 5 mm TBI (triple-broadband inverse) probehead equipped with z-gradient coils was employed for all measurements. IR spectra were measured with the single-reflection ATR (ZnSe) FTIR spectrometer Nicolet Nexus (Thermo, USA). The spectra were acquired by accumulation of 64 scans with 2 cm−1 resolution. 3. Results and discussion 3.1. Identification of unknown impurities The unknown impurities with relative retention time (RRT) 1.38 and 1.50 with respect to RET were obtained in several batches of RET using gradient HPLC method with UV detection at 247 nm (Fig. 1). LC–MS analysis detected ions corresponding to the two compounds, including protonated molecular ion [M+H]+ at m/z = 605.2681 (compound with RRT 1.38) and 605.2680 (compound with RRT 1.50). Use of the HRMS in this measurement allowed determination of the corresponding elemental composition (C32 H34 N6 O4 F2 ) of the both unknown compounds. Moreover, full scan MS spectra and corresponding MS/MS spectra of each compound contained the same molecular ion but different fragments. These findings in the fragmentation pattern hinted the different structure of the corresponding isobaric compounds. Full scan MS spectrum and corresponding MS/MS spectra (at low collision energies) of the compound with RRT 1.38 contained only one dominant signal of the molecular ion, which corresponded to the relatively strong bond between two aromatic rings in the molecule (Fig. 2A). On the other hand, spectra of the compound with RRT 1.50 (recorded at the same experimental conditions as for compound with RRT 1.38) contained two major fragments m/z = 389.1621 (C19 H22 N4 O4 F) and m/z = 302.1301 (C16 H17 N3 O2 F), which can be explained by the connection of the RET monomers through the nitrogen atom (Fig. 2B). The structures of these impurities were verified by NMR analysis after semipreparative isolation.
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Fig. 2. Positive ion mode ESI full scan mass spectra of RET-dimer I (A) and RET-dimer II (B).
The proposed structure of the compound with RRT 1.38 was diethyl {4,4 -diamino-6,6 -bis[(4-fluorobenzyl)amino]biphenyl3,3 -diyl}biscarbamate (RET-dimer I) and the propose structure of the compound with RRT 1.50 was ethyl {2-amino-5-[{2amino-4-[(4-fluorobenzyl)amino]phenyl}(ethoxycarbonyl) amino]-4-[(4-fluorobenzyl)amino]phenyl} carbamate (RET-dimer II). The assignment of NMR signals was performed for both dimeric impurities by means of 1 H, 13 C and 2D (COSY, HSQC and HMBC) spectra. The given structures were unambiguously confirmed (Table 1). If the NMR spectra of the symmetric dimer were confirmed with those of original RET one it was clear that the fluorinated aromatic ring remained untouched and that the two remaining protons (H3 and H6) at the other aromatic ring were in para position to each other. Similarly for nonsymmetric dimer it could be confirmed that the new bond was formed between C4 of one retigabine and N8 of the other. Although the pure RET is a colourless compound, the crude product was intensively coloured. Similarly, RET changed quickly colour from red to dark violet when left on air. This suggests that some quinone diimine structures (supplementary material; supplemental 1) could be involved. Formation of dimers of various substituted 1,4-diaminobenzenes via the corresponding quinone diimines is well demonstrated. The best example is the oxidative selfcoupling of 1,4-diaminobenzene leading to the Bandrowski’s base [10] or reaction of 1,4-diaminobenzene with 1,3-diaminobenzene derivatives under oxidative conditions (supplementary material; supplement 2), which is used also in oxidative hair dye formation
[11]. Since the last steps of the synthesis were done under reductive conditions (supplementary material; supplement 3), these reactions leading to the dimeric product formation could probably take place during work-up and following manipulations. Primarily formed oxidative intermediate could then provide both dimeric impurities. Formation of RET-dimer II, where a C-N bond was formed, was very similar to reactions, which were described in Ref. [10] (supplementary material; supplement 2). On the other hand, formation of RET-dimer I, where a C-C bond was formed, assumed nucleophilic addition of a carbon nucleophile to an activated quinone diimine structure (Fig. 3). Though this type of reaction was not widely reported, some examples were published [12]. Formation of similar dimeric metabolites of flupirtine by similar mechanism involving the quinone diimines has been reported as well [13]. 3.2. Synthesis and characterization of reference standards of impurities 3.2.1. Preparation of RET-dimer I and RET-dimer II Although reductive conditions were used in the final steps of the RET synthesis, the proposed structures of the RET dimeric impurities suggested rather oxidative conditions for their formation. The procedure of synthesis of dimeric impurities was following: A solution of potassium persulfate (K2 S2 O8 , 2.9 g, 10.7 mmol) in 50% aqueous acetonitrile (200 ml) was added during 30 min to a stirred solution of RET (10.0 g, 33.0 mmol) in 50% aqueous acetonitrile (200 ml). The resulting dark purple reaction mixture was stirred
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Table 1 NMR assignments for RET dimers. Position
␦(C)
␦(H)
RET-dimer I 1 2 3 4 5 6 7 8 9 11 12 13 14 = 18 15 = 17 16 20 21
144.9 114.1 131.3 114.7 146.5 99.3 (br.) – – 156.7 – 47.5 137.4 130 116 162.8 61.7 15.1
– – 6.65 – – 5.98 4.04 6.7 – 4.42 4.22 – 7.29 7.02 – 4.08 1.22
RET-dimer II 1 1 2 2 3 3 4 4 5 5 6 6 7; 7 8 9; 9 11; 11 12 12 13; 13 14 = 18; 14 = 18 15 = 17; 15 = 17 16; 16 20; 20 21; 21
143.5 141.6 119.3 118.7 127.7 125.7 103 112.4 147.5 141.6 99.9 97.9 – – 154.5; 154.6 – 45.3 45.7 135.2; 136.6 128.3; 128.5 114.3; 114.4 160.7; 160.8 59.4; 60.7 14.05; 14.10
– – – – 6.76 6.93 5.96 – – – 6.05 5.92 4.45 7.91 – 5.75 4.19 4.21 – 7.18; 7.38 6.99; 7.08 – 4.06; 4.08 1.13; 1.21
Mult.
Integ. H
s
2
s s s (broad)
2 4 2
t d
2 4
m m
4 4
quartet t (broad)
4 6
∼7 ∼7
d s dd
1 1 1
8.5
d s s (broad) s (broad)
1 1 4 1
2.5
t (broad) d d
1; 1 2 2
m; m m; m
2; 2 2; 2
q; q t; t
2; 2 3; 3
Note: RET-dimer I: ␦ in ppm, J in Hz. Reference for 1 H: acetonitrile-d6 (1.94 ppm), for dimethyl sulfoxide-d6 (2.50 ppm), for 13 C: DMSO-d6 (39.50 ppm).
13
J(H.H)
J(C.F)
6.2 6.2 3 8 21 244
8.6; 2.5
6.2 6 3.0; 3.0 8.0; 8.0 21.1; 21.1 242.0; 242.0 7.0; 7.0 7.1; 7.1
C: acetonitrile-d6 (1.39 ppm); RET-dimer II: ␦ in ppm, J in Hz. Reference for 1H:
for further 10 min, then diluted with water (1000 ml) and extracted with ethyl acetate (3× 500 ml). The combined extracts were washed with water (500 ml), dried over magnesium sulfate and evaporated under reduced pressure. The obtained residue (8.6 g) contained 16.1% of RET-dimer I and 15.7% of RET-dimer II. The product was dissolved in acetonitrile to obtain concentration of 100 mg/ml and subsequently purified by semipreparative HPLC. The collection parameters of the semipreparative HPLC method were optimized with respect to high concentration of RET-dimers in the solution of crude sample. The solution of crude sample was injected into the semipreparative column and the fractions of dimeric impurities were repeatedly collected and combined. The combined fractions were evaporated under vacuum at 50 ◦ C to dryness to obtain pure solid dimeric impurity standards.
RET-dimer I of IR spectra were confirmed by following characteristic absorption maxima: (N H) 3470, 3379, 3362, 3317, (C O) 1724, 1699, (C C) 1626, 1443, ␦(N H) 1527, 1505, (C O) + (C F) 1219, (C O) 1149 cm−1 , the main functional groups of RET-dimer II of IR spectra were confirmed by following characteristic absorption maxima: (N H) 3344, (C O) 1686, (C C) 1602, ␦(N H) 1507, (C O) + (C F) 1217, (C O) 1155 cm−1 . Finally, all impurity reference standards of impurities were quantitatively characterized. The potency of the standards was calculated based on the values obtained from determination of impurities (organic, inorganic, water and residual solvents) by applying the principle of mass balance. The potency of RET-dimer I impurity was 99.2% and potency of RET-dimer II impurity was 91.6%.
3.2.2. The characterization reference standards of impurities The structures of prepared dimeric impurities were confirmed by MS and NMR analysis under the conditions described in Section 3.1 and obtained data were in agreement with the data measured for impurities isolated from RET sample. The qualitative characterization of reference standards was also performed using IR measurements (Fig. 4). The main functional groups of
3.3. Optimization of HPLC-UV method The composition and gradient profile of the mobile phase was optimized to achieve the retention factor of RET k ≥ 3.0, symmetry factor AS ≤ 1.5 and resolution between Imp 2 and RET RS ≥ 2.5. The dead volume of column used was determined using injection of sodium nitrate. The chromatographic data were calculated in
M. Douˇsa et al. / Journal of Pharmaceutical and Biomedical Analysis 94 (2014) 71–76
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Fig. 3. Proposed mechanism of formation of dimeric impurities of RET.
agreement with European Pharmacopoeia (Ph.Eur.) [14]. Acetonitrile was selected as sample solvent. The separation of RET and its dimeric impurities was optimized by varying acetonitrile content, concentration and pH of potassium hydrogenphosphate buffer in the mobile phase. All experiments were performed using 10 mM phosphate buffer. The pH was varied in the range from 6.0 to 8.0. Acceptable resolution of tested impurities was achieved with gradient elution of acetonitrile as the organic modifier using flow rate 1.2 ml/min at temperature of 40 ◦ C and 10 mM buffer at pH of 7.5. The chromatogram of separation of RET and its dimeric impurities measured at the optimized HPLC conditions is shown in Fig. 1.
3.4. Validation of HPLC-UV method The method was validated according to ICH Q2(R1) guideline [15]. 3.4.1. System suitability The system suitability test was performed before each run to assure that the analytical method could be used with a satisfactory performance. Repeatability of injections expressed as relative standard deviation (RSD %) of peak area of RET and resolution between Imp 2 and RET for the five consecutive injections of a system
Fig. 4. IR spectra of RET-dimer I (A) and RET-dimer II (B).
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Table 2 The validation parameters of method. Parameter Linearity Slope (V/%) Intercept (V) Correlation coefficient QC coefficient LOD (%) LOQ (%)
4. Conclusion
RET
RET-dimer I
RET-dimer II
289 967 −10.9 0.999 2.0 0.001 0.004
384 404 −37.1 0.999 2.1 0.001 0.003
332 941 86.8 0.999 2.3 0.001 0.003
suitability test solution were limited to ≤1.0% and RS ≥ 2.5 respectively. For all the measurements performed during the validation, the resolution more than 3.0 and RSD of peak areas ≤0.4% was achieved. 3.4.2. Limit of detection (LOD), limit of quantification (LOQ) and linearity LOD and LOQ were calculated for all impurities based on signalto-noise ratio (S/N). The baseline noise was measured in a blank injection in the region of retention time of relevant impurity using chromatographic software. Calculated LOD and LOQ are listed in Table 1. A set of all six impurities and RET solutions at the concentration range from LOQ to 200% of the general specification limit (LOQ – 3.0 g/ml) were prepared. The calibration curves were constructed by plotting the peak area of a given analyte against its concentration. The calibration equations were calculated using linear regression analysis. The parameters of calibration curves are shown in Table 2. The linearity of the calibration curves was investigated using other statistical approaches such as the quality coefficient QC [16]. If the quality coefficient QC fulfilled the criterion QC < 5%, the linearity of calibration model was demonstrated (Table 1). The calculated parameters of calibration curves indicated satisfactory linearity. The correction response factor (CRF) of RET-dimer I and RET-dimer II with respect to RET at 247 nm was obtained from the slope ratio of the appropriate calibration curves. CRF for RET-dimer I was 0.75 and RET-dimer II was 0.83. 3.4.3. Method selectivity No interfering coeluting peaks in blank solvent were observed which demonstrated an adequate selectivity of HPLC method. The selectivity of this method was further evaluated using forced degradation studies. The samples were subjected to hydrolysis (water at 90 ◦ C for 8 h). The PDA detector was used to demonstrate the spectral peak purity of all degradation products. The formation of dimeric impurities, 4-fluorobenzaldehyde, impurity with RRT 0.68 and RRT 0.85 was observed. LC–HRMS high resolution measurement revealed ions corresponding to the unknown impurity with RRT 0.68, including protonated molecular ion [M+H]+ at m/z = 258.1040. Use of the high resolution MS in this measurement allowed determination of the corresponding elemental composition (C14 H12 N3 OF) of the unknown compounds. The proposed structure of this impurity was 5-[(4-fluorobenzyl)amino]-1,3dihydro-2H-benzimidazol-2-one. This impurity was found after the stress test only and was not observed in any of the RET samples. Therefore, this impurity was not characterized anymore. The proposed HPLC method was selective for the determination of process-related impurities in RET.
The new related compounds of RET were found using highly selective HPLC-UV method and identified by means of LC–MS, NMR and IR studies. These impurities were generated in the last step of the RET synthesis and were identified as diethyl {4,4 -diamino-6,6 -bis[(4-fluorobenzyl)amino]biphenyl3,3 -diyl}biscarbamate (RET-dimer I) and ethyl {2-amino-5[{2-amino-4-[(4-fluorobenzyl) amino] phenyl} (ethoxycarbonyl) amino]-4-[(4-fluorobenzyl)amino] phenyl}carbamate (RET-dimer II). The analytical standards of both RET-dimers were synthesized and fully characterized. The quick and efficient HPLC method for the measurement of RET and related-process impurities has been proposed. The method has various advantages over those previously reported, such as very short analysis time and high separation efficiency. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jpba.2014.01.042. References [1] G. Blackburn-Munro, W. Dalby-Brown, N.R. Mirza, J.D. Mikkelsen, R.E. Blackburn-Munro, Retigabine: chemical synthesis to clinical application, CNS Drug Rev. 11 (2005) 1–20. [2] M. Bialer, H.S. White, Key factors in the discovery and development of new antiepileptic drugs, Nature Rev. Drug Discov. 9 (2010) 68–82. [3] C. Rundfeldt, The new anticonvulsant retigabine (D-23129) acts as an opener of K+ channels in neuronal cells, Eur. J. Pharmacol. 336 (1997) 243–249. [4] A. Rostock, C. Tober, C. Rundfeldt, R. Bartsch, J. Engel, E.E. Polymeropoulos, B. Kutscher, W. Löscher, D. Hönack, H.S. White, H.H. Wolf, D-23129: a new anticonvulsant with a broad spectrum activity in animal models of epileptic seizures, Epilepsy Res. 23 (1996) 211–223. [5] A. Hiller, N. Nguyen, C.P. Strassburg, Q. Li, H. Jainta, B. Pechstein, P. Ruus, J. Engel, R.H. Tukey, T. Kronbach, Retigabine N-glucuronidation and its potential role in enterohepatic circulation, Drug Metab. Dispos. 27 (1999) 605–612. [6] G.M. Ferron, J. Paul, R. Fruncillo, L. Richards, N. Knebel, J. Getsy, S. Troy, Multiple-dose linear, dose-proportional pharmacokinetics of retigabine in healthy volunteers, J. Clin. Pharmacol. 42 (2002) 175–182. [7] W. Bu, M. Nguyen, C. Xu, C.C. Lin, L.T. Yeh, V. Borges, Determination of N-acetyl retigabine in dog plasma by LC/MS/MS following off-line microelution 96-well solid phase extraction, J. Chromatogr. B 852 (2007) 465–472. [8] N.G. Knebel, S. Grieb, S. Leisenheimer, M. Locher, Determination of retigabine and its acetyl metabolite in biological matrices by on-line solid-phase extraction (column switching) liquid chromatography with tandem mass spectrometry, J. Chromatogr. B 748 (2000) 97–111. [9] X. Wang, H. Zhou, J. Zheng, C. Huang, W. Liu, L. Yu, S. Zeng, Identification and characterization of four process-related impurities in retigabine, J. Pharm. Biomed. Anal. 71 (2012) 148–151. [10] E.A. Shilova, A. Heynderickx, O. Siri, Bandrowski’s base revisited: toward an unprecedented class of quinonediimines or new two-way chromophoric molecular switches, J. Org. Chem. 75 (2010) 1855–1861. [11] Scientific Committee on Consumer Safety SCCP/0941/05, in: Opinion on Exposure to Reactants and Reaction Products of Oxidative Hair Dye Formulations, 2005. [12] D.L. Boger, H. Zarrinmayeh, Regiocontrolled nucleophilic addition to selectively activated p-quinone diimines: alternative preparation of a key intermediate employed in the preparation of the CC-1065 left-hand subunit, J. Org. Chem. 55 (1990) 1379–1390. [13] K. Methling, P. Reszka, M. Lalk, O. Vrana, E. Scheuch, W. Siegmund, B. Terhaag, P.J. Bednarski, Drug Metab. Disp. 37 (2009) 479–493. [14] European Pharmacopoeia, Strasbourg: Council of Europe, 6th ed., 2009, pp. 4407–4413. [15] ICH Q2(R1), Validation of Analytical Procedures; Step 4 Version, 2005. [16] J. Van Loco, M. Elskens, Ch. Croux, H. Beernaert, Linearity of calibration curves: use and misuse of the correlation coefficient, Accredit. Qual. Assur. 7 (2002) 281–285.
