Journal of Pharmaceutical and Biomedical Analysis 95 (2014) 256–264

Contents lists available at ScienceDirect

Journal of Pharmaceutical and Biomedical Analysis journal homepage: www.elsevier.com/locate/jpba

Identification and characterization of stress degradants of lacosamide by LC–MS and ESI-Q-TOF-MS/MS: Development and validation of a stability indicating RP-HPLC method Nageswara Rao Ramisetti a,∗ , Ramakrishna Kuntamukkala a , Sridhar Lakshetti b , Prabhakar Sripadi b a b

HPLC Group, Analytical Chemistry Division, IICT, Tarnaka, Hyderabad 500007, India National Centre for Mass Spectrometry, IICT, Tarnaka, Hyderabad 500007, India

a r t i c l e

i n f o

Article history: Received 18 September 2013 Received in revised form 4 March 2014 Accepted 7 March 2014 Available online 15 March 2014 Keywords: ESI-MS/MS Forced degradation Lacosamide RP-HPLC Stability indicating

a b s t r a c t The current study dealt with the degradation behavior of lacosamide (LAC) under ICH prescribed stress conditions. LAC was found to be labile under acid and base hydrolytic stress conditions, while it was stable to neutral hydrolytic, oxidative, photolytic and thermal stress. In total, seven degradation products (DPs) were formed, which were separated on a C18 column using a stability-indicating method. LC–MS analyses indicated that one of the DPs had the same molecular mass as that of the drug. Structural characterization of DPs was carried out using ESI-Q-TOF-MS/MS technique. The degradation pathways and mechanisms of degradation of the drug were delineated by carrying out the degradation in different co-solvents viz. methanol, deuterated methanol, ethanol, 1-propanol and acetonitrile. The developed LC method was validated for the determination of related substances and assay of LAC as per ICH guidelines. This study demonstrates a comprehensive approach of LAC degradation studies during its development phase. © 2014 Published by Elsevier B.V.

1. Introduction Lacosamide (LAC), N2 -acetyl-N-benzyl-d-homoserinamide, is a novel antiepileptic drug approved by the U.S. Food and Drug Administration (FDA) for the adjunct treatment of partial onset seizures in patients with and without secondary generalization by selectively modulating the voltage-gated sodium channels by enhancing slow inactivation without affecting fast inactivation to exert its antiepileptic activity [1–3]. Stability testing of drug substances is a part of development strategy and is normally carried out under more severe conditions compared to accelerated stress testing. Its aim is to provide evidence on how the quality of a drug substance varies with time under the influence of various environmental conditions such as temperature, humidity, pH, and light, which enables in recommending the preferred storage conditions,

∗ Corresponding author. Tel.: +91 40 27193193; fax: +91 40 27160387. E-mail addresses: [email protected], [email protected] (N.R. Ramisetti). http://dx.doi.org/10.1016/j.jpba.2014.03.010 0731-7085/© 2014 Published by Elsevier B.V.

retest periods and establishing its shelf life. The content of related substances (RSs) and assay of LAC bulk drugs is required to be determined using stability indicating method and characterization of potential process RSs and degradation products (DPs) in the quality control of LAC bulk drugs is much needed [4–8]. A few chromatographic methods have been published in the literature for the quantification of LAC including the analysis of LAC in rat and human plasma by LC–MS/MS and HPLC–UV respectively [9,10]. Stability-indicating HPLC methods for the determination of LAC were also reported [11–13]. But none of the methods describes the characterization of all DPs and the analysis of LAC in the presence of both process RSs as well as DPs formed under stress conditions. Recently, Tiwari and Bonde have identified and characterized two degradation products of LAC by liquid-chromatography/time-of-flight mass spectrometric and multi-stage mass spectrometry [14]. However, the data was wrongly interpreted and characterization of one of the degradation products was incorrect. To the best of our knowledge, yet no stability-indicating LC method for the quantitative estimation of LAC drug substance in the presence of its RSs and characterization

N.R. Ramisetti et al. / Journal of Pharmaceutical and Biomedical Analysis 95 (2014) 256–264

of all the DPs have been reported. The purpose of the present work was to develop a stability-indicating LC method for the determination of LAC and its RSs and the characterization of DPs by LC–MS and ESI-Q-TOF-MS/MS. 2. Materials and methods 2.1. Chemicals Samples and standards (purity > 99%) of LAC and its process RSs (RS1: benzyl amine, RS2: 2-acetamido-N-benzyl-3hydroxypropanamide) were obtained from local industry, Hyderabad, India. HPLC pure water (Milli-Q water purification system, Millipore synergy, France), HPLC grade MeOH (Sigma Aldrich, India), AR grade KH2 PO4 , HCOONH4 , H3 PO4 , and HCOOH (S.D. Fine Chem., Mumbai, India) were used. Mixture of H2 O and MeOH (40:60) was used as diluent. 2.2. Instrumentation LC-PDA and LC–MS experiments were carried out on a Shimadzu prominence HPLC system equipped with a quaternary UFLC LC-20AD pump, a DGU-20A5 degasser unit, a SPD-M20A diode array detector, quadrupole mass spectrometer equipped with an ESI source, a SIL-20AC auto sampler, a CTO-20AC column oven and CBM-20A communications bus module was used for method development and validation studies. The data acquisition and processing were carried out using Lab Solutions software. Accurate mass measurements and MS/MS studies were carried out by using a quadrupole time-of-flight (Q-TOF) mass spectrometer (QSTAR XL; Applied Biosystems/MDS Sciex, Foster City, CA, USA), equipped with an ESI source. The data acquisition was under the control of Analyst QS software (Foster City, CA). 2.3. High performance liquid chromatography conditions A Kinetex C18 column (150 mm × 4.6 mm; 5 ␮) was used with the mobile phase consisting of KH2 PO4 (pH 4.5, 0.02 M) (solvent A), and MeOH (solvent B) in a gradient elution mode pumped through the column at a flow rate of 1.0 ml/min at column temperature of 25 ◦ C. The sample injection volume was 10 ␮L. The gradient program was time (min)/% B: 0/20, 12/20, 15/40, 17/40, 17.1/20, 26/20. Eluents were monitored at 210 nm. For the identification of DPs by LC–MS, phosphate buffer was replaced with HCOONH4 (pH 4.5 adjusted with HCOOH, 0.02 M) without changing the other conditions. Semi-preparative isolation of LAC isomeric degradation product (DP6) was carried out with mobile phase consisting of H2 O and MeOH (70:30, v/v) on an Agilent XDB-C18 (250 mm × 9.0 mm, 5 ␮). 2.4. Mass spectrometry conditions for LC–MS Nitrogen was used as the nebulizing and dissolving gas at a flow rate of 50 and 400 Lit/h, respectively. The optimum source (ESI) conditions were: spray voltage 3.5 kV, cone voltage 25 V, extractor 3 V, rf lens 0.2 V, source temperature 100 ◦ C, ESI probe temperature 300 ◦ C. 2.5. Mass spectrometry conditions for MS/MS All the samples were introduced directly into the source by flow injection (10 ␮l loop) method using methanol as the mobile phase at a flow rate of 0.3 ml/min. Typical source conditions were: capillary voltage, 5 kV; declustering potentials (1) 60 V; and (2) 10 V; focusing potential, 250 V; mass resolution 10,000 (FWHM). Ultra-high-purity nitrogen was used as the curtain gas and the

257

collision gas, and zero air was used as the nebulizer gas. For the CID experiments, the precursor ion, [M+H]+ was selected using the quadrupole analyzer and the product ions were analyzed using the TOF analyzer. The collision energies used were 10–25 eV. All the spectra reported were averages of 25–30 scans. The elemental compositions for the precursor as well as product ions were obtained from the measured accurate mass values by using the Analyst QS software. 2.6. Preparation of analytical solutions Stock solutions of LAC (2.0 mg/ml) and each RS (0.5 mg/ml) were prepared by dissolving the appropriate amounts in the diluent. Working solutions were prepared by adequately mixing the stock solutions with diluent. 2.7. Specificity and forced degradation Specificity is the ability of the method to measure the LAC response in the presence of its potential process RSs and DPs. Stress testing of the drug substance can help to identify the possible DPs, which in turn can help to establish the degradation pathways and the intrinsic stability of the molecule. The specificity of the developed LC method for LAC was carried out in the presence of its process RSs and DPs. LAC was subjected to stress degradation such as acid (2 N HCl, 14 h), alkaline (1 N NaOH, 2 h), neutral hydrolysis (24 h) and oxidation (14% H2 O2 , 24 h) conditions. Sample was also exposed to heat (60 ◦ C, 7 days) and UV light (254 nm, deuterium lamp, 7 days) degradation in dry state (≈1 mm thickness) in petri dishes. Methanol, deuterated methanol, ethanol, 1-propanol and acetonitrile were used as co-solvents to predict the possible pathway of hydrolytic degradation of LAC. Different stress conditions were followed to achieve significant degradation. The collected degradation samples were neutralized (for acidic and basic hydrolyzed) and analyzed by LC-PDA for the determination of the total impurities and assay of LAC. LC–MS and ESI-MS/MS analyses were carried out for the identification and characterization of DPs, respectively. A schematic representation of degradation of LAC is shown in Fig. 1. 2.8. Method validation The proposed HPLC method has been validated as per ICH guidelines [15]. 2.8.1. Limits of detection and quantitation The limits of detection (LOD) and limits of quantitation (LOQ) for all analytes were calculated with a signal-to-noise ratio of 3:1 and 10:1, respectively. LOD and LOQ were determined by injecting a series of diluted solutions of known concentrations of LAC and RSs. 2.8.2. Precision and accuracy The system precision was evaluated by analyzing six replicates of standard solution for both assay (LAC 100 ␮g/ml) and RSs (LAC (1000 ␮g/ml) spiked with 0.15% each RS) individually. The method precisions for assay and RSs were evaluated by injecting six individual test preparations of LAC (100 ␮g/mL), and LAC (1000 ␮g/mL) spiked with 0.15% each RS, respectively. The intermediate precisions were evaluated on a different day by different analyst using different batch column and instrument located within the same laboratory. Precision at LOQ levels was also determined by injecting six individual preparations of mixture of all RSs spiked to LAC (1000 ␮g/ml) at their LOQ level. The % RSDs of the areas of each RS and assay of LAC were calculated for precision studies. For the determination of accuracy of RSs, known amounts of RSs were spiked in

258

N.R. Ramisetti et al. / Journal of Pharmaceutical and Biomedical Analysis 95 (2014) 256–264

Fig. 1. A schematic representation of LAC degradation.

triplicate at three different concentration levels (0.075, 0.15, and 0.225%) to 1000 ␮g/ml of LAC. The accuracy of the LAC assay was evaluated in triplicate at three concentration levels (50, 100, and 150 ␮g/ml). The percentages of recoveries were calculated at each level.

2.8.3. Linearity Linearity of RSs were established by analyzing series of dilute solutions at six different levels ranging from LOQ to 250% (i.e., LOQ, 0.075, 0.15, 0.225, 0.30 and 0.375%) to the specification level of LAC (1000 ␮g/ml). The calibration curves were drawn by plotting

the peak areas of RSs against their corresponding concentrations. Similarly, assay linearity was established by injecting LAC at five concentration levels i.e., 50, 75, 100, 125, and 150%.

2.8.4. Robustness The robustness study was carried out to evaluate the influence of small variations in the optimized chromatographic conditions. The factors chosen for this study were temperature of the column (25 ± 2 ◦ C), mobile phase pH (4.5 ± 0.1), mobile phase composition (initial concentration of organic modifier concentration in gradient program 20 ± 1), buffer strength (0.02 ± 0.002 M) and mobile

Fig. 2. LC-PDA chromatograms of (a) LAC (1000 ␮g/ml) spiked with 1% impurities, (b) LAC acid stressed sample and (c) LAC base stressed sample.

N.R. Ramisetti et al. / Journal of Pharmaceutical and Biomedical Analysis 95 (2014) 256–264

259

Table 1 Validation data. Parameter System suitabilitya RT (min) Rs kl As N

RS1

RS2

LAC

2.3 0 0 0.96 1246

6.7 13.90 1.89 1.17 5352

13.3 13.98 4.76 1.12 9651

Sensitivityb LOD (␮g/ml) LOQ (␮g/ml)

0.03 0.09

0.05 0.15

0.08 0.2

Precision (%RSD)a System Method Intermediate LOQ

0.64 1.56 1.71 2.07

1.13 1.90 2.68 3.15

0.21 0.18 0.13 –

0.09–3.75 0.9999 24,807 −46.75

0.15–3.75 0.9989 24,606 44.97

50–150 0.9998 23,328 16,881

Accuracy at 50% levelb Amount added (␮g/ml) Amount recovered (␮g/ml) % Recovery

0.75 0.751 100.13

0.75 0.727 96.87

50 50.25 100.5

Accuracy at 100% levelb Amount added (␮g/ml) Amount recovered (␮g/ml) % Recovery

1.5 1.475 98.30

1.5 1.466 97.71

100 99.81 99.81

Accuracy at 150% levelb Amount added (␮g/ml) Amount recovered (␮g/ml) % Recovery

2.25 2.200 97.77

2.25 2.277 101.18

150 149.48 99.65

Accuracy at LOQ levelb Amount added (␮g/ml) Amount recovered (␮g/ml) % Recovery

0.09 0.091 101.52

0.15 0.155 103.48

– – –

Linearity Range (␮g/ml) r2 Slope Intercept

RT, retention time; Rs, resolution; kl , retention factor; As , tailing factor; N, number of theoretical plates; r2 , correlation coefficient. a Average of six determinations. b Average of three determinations.

phase flow rate (1.0 ± 0.1 ml/min). System suitability parameters of all analytes and assay of LAC were checked. In the all above varied conditions, the components of the mobile phase were held constant. 3. Results and discussion

good peak parameters compared to acetate and formate buffers at same pH, in which the base line drift was more. Finally MeOH, and KH2 PO4 (pH 4.5, 0.02 M) were used as the mobile phase components in a gradient elution program for HPLC analysis. Same gradient elution program with HCOONH4 buffer (pH 4.5, 0.02 M) and MeOH was used for the identification of DPs by LC–MS. The chromatograms under optimized conditions were shown in Fig. 2.

3.1. Method development and optimization of chromatographic conditions

3.2. Method Validation

The chromatographic conditions were optimized to develop a selective stability-indicating method to separate LAC from its process RSs and DPs. The mobile phases chosen were aqueous buffers (CH3 COONH4 , HCOONH4 and KH2 PO4 ) at different pH, MeOH, and MeCN. Different types of C18 columns (Agilent XDB, Phenomenex Extend, and Kinetex) were studied by subjecting RSs spiked solution of LAC and degradation samples. Finally, after several attempts Kinetex C18 column was found to be better selective compared to other columns with mobile phase consisting of MeOH and phosphate buffer. In the initial trials with higher MeOH content LAC and one of the degradants (DP5) was co-eluting; while at lower content better selectivity was observed for all analytes but with very higher retention times. In order to get better selectivity and with less run time, initially lower MeOH content was maintained and later increased to higher levels. It was observed that phosphate buffer at pH 4.5 (without adjusting) showed better selectivity with

The system suitability and selectivity were conducted by injecting 1000 ␮g/ml of LAC solution containing 1.5 ␮g/ml (0.15%) of all RSs. System suitability results are given in Table 1. The system was deemed to be suitable for use as the tailing factor for all the components was less than 1.2 and the resolution between any of the two analytes was greater than 13. It also confirms the good selectivity of the method. The LOD and LOQ results were obtained in the range of 0.03–0.2 ␮g/ml indicating the higher detection sensitivity of the method. %RSD values of precision experiments were obtained below 3.2 indicates good precision of the method. The percentage recovery range for all related substances (96.87–103.48) and LAC (99.65–100.50) were suitable for the quantitative HPLC method. The peak area versus concentration data was analyzed with least squares linear regression. The results of linearity studies demonstrated good linearity for LAC (r2 = 0.9998) and RSs (r2 ≥ 0.9989). In

260

N.R. Ramisetti et al. / Journal of Pharmaceutical and Biomedical Analysis 95 (2014) 256–264

Table 2 Elemental compositions of [M+H]+ ions and fragment ions in MS/MS spectra of [M+H]+ ions of LAC (m/z 251), DP1 (m/z 108), DP2 (m/z 209), DP3 (m/z 237), DP4 (m/z 177), DP5 (m/z 219) and DP6 (m/z 251). Analyte (RT min)

Proposed formula

Observed mass (Da)

Calculated mass (Da)

Error (ppm)

Proposed neutral loss

LAC (13.3)

C13 H19 N2 O3 C12 H15 N2 O2 C11 H17 N2 O2 C10 H13 N2 O C6 H10 NO3 C5 H10 NO2 C7 H10 N C7 H7

251.1395 219.1126 209.1286 177.1028 144.0661 116.0712 108.0813 91.0548

251.1390 219.1128 209.1290 177.1027 144.0655 116.0706 108.0813 91.0547

−1.99 0.91 1.91 −0.56 −4.16 −5.16 0 −1.09

– CH4 O C2 H2 O C3 H6 O2 C7 H9 N C8 H9 NO C6 H9 NO3 C6 H12 N2 O2

DP1 (2.3)

C7 H10 N C7 H7

108.0818 91.0545

108.0813 91.0547

−4.62 2.19

C6 H9 NO3 NH3

DP2 (5.7)

C11 H17 N2 O2 C10 H13 N2 O C10 H13 NO C9 H10 NO C7 H10 N C7 H7 C3 H8 NO C2 H6 N

209.1296 177.1026 164.1076 148.0754 108.0808 91.0543 74.0610 44.0499

209.1290 177.1027 164.1075 148.0762 108.0813 91.0547 74.0605 44.0495

−2.86 0.56 −0.6 5.4 4.62 4.39 −6.75 −9.0

C2 H2 O CH4 O CH4 NO C2 H7 NO C4 H7 NO2 C4 H10 N2 O2 C8 H9 NO C9 H11 NO2

DP3 (6.7)

C12 H17 N2 O3 C12 H15 N2 O2 C10 H12 NO2 C10 H13 N2 O C8 H10 NO C7 H10 N C7 H7

237.1242 219.1126 178.0874 177.1017 136.0752 108.0808 91.0546

237.1239 219.1128 178.0868 177.1027 136.0762 108.0813 91.0547

−1.26 0.91 −3.36 5.6 7.3 4.62 1.09

CH2 H2 O C2 H5 NO C2 H4 O2 C4 H7 NO2 C5 H7 NO3 C5 H10 N2 O3

DP4 (7.6)

C10 H13 N2 O C10 H10 NO C10 H11 N2 C9 H10 N C7 H7 C4 H4 NO

177.1034 160.0767 159.0907 132.0815 91.0551 82.0288

177.1027 160.0762 159.0922 132.0813 91.0547 82.0292

−3.95 −3.12 9.42 −1.51 −4.39 4.87

C3 H6 O2 NH3 H2 O CH3 NO C3 H6 N2 O C6 H9 N

DP5 (14.4)

C12 H15 N2 O2 C12 H13 N2 O C10 H13 N2 O C10 H10 NO C5 H6 NO2 C7 H10 N C7 H7 C4 H4 NO

219.1126 201.1014 177.1026 160.0767 112.0387 108.0806 91.0546 82.0291

219.1128 201.1022 177.1027 160.0762 112.0398 108.0813 91.0547 82.0292

0.91 3.97 0.56 −3.12 9.81 6.47 1.09 1.21

CH4 O H2 O C2 H2 O C2 H5 NO C7 H9 N C5 H5 NO2 C5 H8 N2 O2 C8 H11 NO

DP6 (17.2)

C13 H19 N2 O3 C12 H15 N2 O2 C11 H14 NO2 C10 H13 N2 O C7 H10 N C7 H7

251.1394 219.1126 192.1036 177.1020 108.0812 91.0546

251.1390 219.1128 192.1025 177.1027 108.0813 91.0547

−1.59 0.91 −5.72 3.95 0.92 1.09

– CH4 O C2 H5 NO C3 H6 O2 C6 H9 NO3 C6 H12 N2 O2

RT, retention time.

all varied chromatographic conditions (i.e., column temperature, mobile phase composition, pH, flow rate, and buffer strength) of robustness study, the resolution between any of two analytes was found to be greater than 9.0, tailing factor of each analyte was obtained below 1.4, and assay of LAC obtaining in the range of 98.4–101.3 illustrating the robustness of the method. The results of LOD, LOQ, precision, accuracy and linearity are summarized in Table 1.

99.0%. Summary of forced degradation studies of LAC consisting of assay, total impurities and mass balance is given in supporting information (Table S3). All the DPs formed were well separated from LAC and each other (Rs > 2), the assay of LAC was also found unaffected in the presence of RSs mixture confirming the specificity, selectivity and stability-indicating power of the developed method. 3.4. Characterization of LAC and its DPs by LC–MS and MS/MS

3.3. Degradation behavior of LAC Degradation of LAC was not observed in neutral hydrolysis, oxidation, light and heat. Significant degradation of LAC was observed in acid (15%) and base (20%) hydrolytic conditions. Three DPs were formed in acid (DP1, DP2, and DP3) and in addition, four more DPs (UK (unknown), DP4 DP5, and DP6) were formed in base (Fig. 2). Peak purity test results derived from the PDA detector, confirmed that the LAC peak was homogeneous and pure in all analyzed stress samples. The mass balance of the stressed samples was close to

LAC and all the DPs (DP1–DP6) were well separated by LC–ESIMS and their retention times (RT) were given in Table 2. All the compounds showed abundant protonated molecular ions, under positive ESI conditions. Collision induced dissociation (CID) spectra of the [M+H]+ were recorded to obtain structure information. For CID experiments, the samples were directly infused into the ESI source. The isomeric compounds, LAC and DP6 were isolated from the reaction mixture using semi-preparative HPLC, before subjecting to CID experiments. The fragmentation patterns were arrived

N.R. Ramisetti et al. / Journal of Pharmaceutical and Biomedical Analysis 95 (2014) 256–264

261

Fig. 3. ESI-MS/MS spectra of [M+H]+ ions of LAC (m/z 251), DP1 (m/z 108), DP2 (m/z 209), DP3 (m/z 237), DP4 (m/z 177), DP5 (m/z 219) and DP6 (m/z 251).

based on MS/MS experiments and accurate mass measurements from high resolution mass spectral (HRMS) data. The ESI-MS/MS spectra of LAC and all the DPs and the fragmentation patterns of their [M+H]+ ions were shown in Figs. 3 and 4, respectively. The elemental composition of [M+H]+ ions of all the DPs and their fragment ions obtained from HRMS data were summarized in Table 2.

3.4.1. MS/MS of LAC The positive ion ESI-MS of LAC shows abundant [M+H]+ ion at m/z 251. The ESI-MS/MS spectrum of [M+H]+ ion of LAC showed abundant product ions at m/z 108 corresponds to protonated benzyl amine, which further fragmented to yield a tropylium cation

(m/z 91) by the loss of NH3 . These two product ions confirm the presence of the benzyl amine group in the molecule. The spectrum also include abundant product ions at m/z 219, 177 and 144 due to the loss of CH3 OH, (CH3 OH+CH2 CO) and (C6 H5 CH2 NH2 ), respectively, from [M+H]+ ion. In addition, the spectrum showed low abundant product ions corresponding to [MH−CH2 CO]+ (m/z 209), [MH−CH3 OH+CH2 CO−NH3 ]+ (m/z 160), [MH−C6 H5 CH2 NH2 −CO]+ (m/z 116), and the benzyl cation (m/z 91) (Figs. 3 and 4).

3.4.2. DP1 (m/z 108) The ESI-MS/MS spectrum of the ion at m/z 108 (DP1) showed the abundant product ion at m/z 91, a tropylium cation, which was

262

N.R. Ramisetti et al. / Journal of Pharmaceutical and Biomedical Analysis 95 (2014) 256–264

Fig. 4. Proposed fragmentation mechanisms of [M+H]+ ions of (a) LAC, (b) DP2, (c) DP3, (d) DP4, (e) DP5, and (f) DP6.

characteristic of a benzyl group. The ion m/z 91 is formed by the loss of 17 Da (NH3 ) indicating the presence of a primary amine (NH2 ) group. Thus, formation of m/z 91 by the loss of ammonia confirms that the ion m/z 108 corresponds to protonated benzyl amine (DP1). The elemental composition of the parent and product ions confirms the proposed structure for DP1 (Table 2), which is one of the process RSs (RS1) of LAC.

3.4.3. DP2 (m/z 209) The ESI-MS/MS spectrum of [M+H]+ ion (m/z 209) of DP2 displayed a low abundant, but characteristic [MH−NH3 ]+ ion at m/z 192, which indicates the presence of a free –NH2 group. The spectrum included abundant product ions at m/z 164 (loss of CO from m/z 192), m/z 108 (protonated benzyl amine), m/z 91 (tropylium cation), m/z 102 (loss of benzyl amine from [M+H]+ ion) and m/z 74 (2-methoxy ethanaminium ion). In addition, the spectrum also showed low abundant ions at m/z 177 (loss of CH3 OH from m/z 209), m/z 148 (loss of CH4 from m/z 164), 120 (loss of CO from m/z 148), m/z 83 (loss NH3 from m/z 102) and m/z 44 (loss of CH2 O from m/z 74) (Figs. 3 and 4 and Table 2). The ion at m/z 177 confirms presence of a methoxy group and the ion at m/z 102 and its further fragmentation confirms the presence of 3-methoxy, 2-amino propionyl group (H3 C–O–CH2 –CH(NH2 )–CO–) in the structure of DP2.

Absence of a ketene loss from [M+H]+ ion indicates lack of an acetyl group in DP2. Based on the above discussed fragmentation pattern, DP2 was identified as 2-amino-N-benzyl-3-methoxypropanamide.

3.4.4. DP3 (m/z 237) The ESI-MS/MS spectrum of the ion m/z 237 corresponding to the [M+H]+ of DP3, showed a low abundant but characteristic ion at m/z 219 corresponding to [MH−H2 O]+ . The spectrum include abundant product ions at m/z 177 and 178 corresponding to [MH−H2 O−CH2 CO]+ and [MH−CH3 CONH2 ]+ ion, respectively. The spectrum also include the ions at m/z 108 (protonated benzyl amine), m/z 91 (tropylium cation) and m/z 60 (protonated acetamide). In addition, the spectrum also showed low abundance ions at m/z 160 (loss of NH3 from m/z 177), m/z 132 (loss of CO from ion m/z 160), m/z 112 (loss of benzyl amine from m/z 219) and m/z 201 (loss of H2 O from the ion m/z 219) (Figs. 3 and 4 and Table 2). The formation of ions at m/z 108, m/z 91 indicates the presence of a benzyl amine group in the structure of DP3. The ion at m/z 178 indicates the presence of a CH3 –CO–NH– group in DP3. Loss of two consecutive water molecules from [M+H]+ ion suggest that the structure contains at least one free –OH group (being other expected from the amide bond as found in DP5). Therefore, DP3

N.R. Ramisetti et al. / Journal of Pharmaceutical and Biomedical Analysis 95 (2014) 256–264

263

was identified as 2-acetamido-N-benzyl-3-hydroxypropanamide, which is one of the process RSs (RS2) of LAC. 3.4.5. DP4 (m/z 177) The ESI-MS/MS spectrum of the ion at m/z 177, [M+H]+ ion of DP4 showed a major product ion at m/z 91 corresponds to the tropylium cation. In addition, the spectrum included low abundance ions at m/z 160 (loss of NH3 ), 159 (loss of H2 O), and 132 (loss of NH3 +CO) from [M+H]+ ion (Figs. 3 and 4 and Table 2). Loss of NH3 molecule from [M+H]+ ion indicates the presence of free –NH2 group and the absence of ketene (–CH2 CO) loss indicates that there is no acetyl group present in DP4. Based on the above discussed fragmentation pattern, DP4 was identified as 2-amino-N-benzylacrylamide. 3.4.6. DP5 (m/z 219) The ESI-MS/MS spectrum of [M+H]+ ion of DP5 (m/z 219) showed abundant product ions at m/z 177 corresponding to [MH–CH2 CO]+ and m/z 108 corresponding to protonated benzyl amine. In addition, the spectrum also showed low abundant ions at m/z 201 [loss of H2 O from (M+H)+ ], m/z 160 (loss of NH3 from m/z 177), m/z 112 (loss of benzyl amine from [M+H]+ ), and m/z 91 (tropylium cation) (Figs. 3 and 4 and Table 2). Formation of [MH-H2 O]+ ion could be explained by initial keto-enol tautomerism of one of the amide bond. The loss of CH2 CO followed by NH3 from [M+H]+ ion indicates the presence of an acetamide group (CH3 –CO–NH–) in the structure of DP5. The formation of ions at m/z 108 and 91 indicates the presence of benzyl amine group and the ion at m/z 112 indicates other part of the structure of DP5. Therefore, DP5 was identified as 2-acetamido-N-benzylacrylamide. 3.4.7. DP6 (m/z 251) The ESI-MS/MS spectrum of [M+H]+ ion (m/z 251) displayed abundant product ions at m/z 219 corresponding to the loss of CH3 OH from [M+H]+ , which reveals the presence of a –OCH3 group in the molecule. Further loss of a CH2 CO from m/z 219 to yield the ion at m/z 177 confirmed the presence of an acetyl group in the molecule. The ion at m/z 108 (protonated benzyl amine) and m/z 91 confirmed the presence of benzyl amino group. In addition, the spectrum also showed low abundance ion at m/z 192 (loss of CH3 CONH2 from m/z 251) and m/z 160 (loss of NH3 from the ion m/z 177) (Figs. 3 and 4 and Table 2). Therefore, DP6 was identified as 2-acetamido-N-benzyl-2-methoxypropanamide. 3.5. Differentiation of LAC & DP6 LAC and DP6 were isomeric in nature. It was observed that LAC and DP6 showed a few differences in their fragmentation pattern of [M+H]+ ions, though there were some common product ions. The CID of [M+H]+ ion of LAC showed specific product ions [MH–C6 H5 CH2 NH2 ]+ (m/z 144) and [MH–C6 H5 CH2 NH2 –CO]+ (m/z 116), which were absent in the case of DP6. The ion at m/z 108 (protonated benzyl amine) was the base peak in LAC, while it was low abundant (12%) in the case of DP6. Likewise, the ion at m/z 91 was relatively more abundant (6%) in LAC than in DP6 (0.3%). The [MH–CH3 OH]+ ion (m/z 219) is selectively abundant in DP6 (100%) when compared to that in LAC (29%); this selectivity may be due to attachment of the methoxy group to a tertiary carbon in DP6. The ion [MH–CH3 CONH2 ]+ (m/z 192) was found to be specific for DP6. Based on the differences found in the CID mass spectra, the isomeric compounds can be easily differentiated from one another. 3.6. Characterization of isomer of LAC by NMR One of the DPs (DP6) formed under methanolic basic stress possess same m/z as that of LAC. To characterize the DP6, it was isolated by semi-preparative HPLC and subjected for NMR spectral

Fig. 5. ESI-MS spectra of LAC base stressed samples in CD3 OD, EtOH and 1-PrOH.

studies. LAC and DP6 were assigned by 1 H and 13 C NMR in CDCl3 at 300 MHz and 75 MHz, respectively. The formation of positional isomer of LAC during its degradation was confirmed by 2D NMR studies such as double quantum filtered correlation spectroscopy (DQF-COSY) in which the individual spin systems were identified and 1 H–13 C HSQC experiments in which the carbon resonance was identified via proton observation. The COSY spectrum of LAC showed the correlation of H-4 with H-3 and H-8a and H-8b, H-6 with H-7 revealed the confirmation of LAC; whereas, DP6 showed the correlation of H-6 with H-7 confirmed it as a positional isomer of LAC. The NMR data and spectra of LAC and DP6 are given in supporting information (Tables S1 and S2; Figs. S1 and S2). 3.7. Mechanism of formation of isomer (DP6) and effect of co-solvent on hydrolysis of LAC To study the mechanism of formation of DP6, an isomer of LAC, hydrolysis experiments were conducted in different co-solvents viz. MeOH, CD3 OD, EtOH, 1-PrOH, and MeCN. The isomer DP6 was formed when LAC was subjected to base hydrolysis in the presence of MeOH. Its ESI mass spectrum has shown the [M+H]+ and [M+Na]+ ions at m/z 251 and m/z 273, respectively. When the same reaction was performed in the presence of EtOH the above ions shifted 14 u, where the [M+H]+ ion appeared at m/z 265 and [M+Na]+ at m/z 287. Similarly, when the reaction is performed in the presence of 1-PrOH, the ions were shifted 28 u, appearing [M+H]+ ion at m/z 279 and [M+Na]+ at m/z 301 (Fig. 5). When MeOH was replaced with CD3 OD, as expected, there was a shift of 3 u; the [M+H]+ and [M+Na]+ ions were appeared at m/z 254 and 276, respectively. The DP6 was not formed when MeCN was used as co-solvent, as confirmed by LC–MS analysis. The ions due to other DPs were found to be intact in all the above reactions. All these experimental results clearly confirms that the DP6 is formed by the addition of co-solvent (alcohol) to the DP5, which is a major DP formed by the elimination

264

N.R. Ramisetti et al. / Journal of Pharmaceutical and Biomedical Analysis 95 (2014) 256–264

of MeOH from LAC. Effect of co-solvent was also tested for acid hydrolysis reaction and found that there were no changes, because the major degradation process was deacetylation (DP2). 4. Conclusions The forced degradation behavior of lacosamide (LAC) was studied as per ICH guidelines. Seven degradation products (DPs) were formed under stress conditions as detected by HPLC, LC–MS, and high resolution ESI-MS. The proposed structures for the DPs were supported by high resolution ESI-MS/MS analysis and accurate mass measurements. A simple gradient RP-HPLC method has been developed and validated for the determination of stability indicating assay of LAC and its related substances in bulk drugs. The developed method has been found to be selective, accurate, sensitive and precise, and is applicable for detecting process-related substances and other possible degradants which may be present at trace level in bulk drugs. NMR and ESI-MS techniques were successfully used to study the effect co-solvent on hydrolysis of LAC as well as to confirm the formation of DP6, which is isomeric to LAC, during methanolic basic stress. Acknowledgements The authors thank Director, IICT, Hyderabad, India for encouragement and permission to communicate results for publication. RK and SL thank CSIR and UGC, New Delhi, respectively, for Research Fellowships. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jpba.2014.03.010. References [1] C. Kellinghaus, Lacosamide as treatment for partial epilepsy: mechanisms of action, pharmacology, effects, and safety, Ther. Clin. Risk. Manag. 5 (2009) 757–766.

[2] G. Curia, G. Biagini, E. Perucca, M. Avoli, Lacosamide: a new approach to target voltage-gated sodium currents in epileptic disorders, CNS Drugs 23 (2009) 555–568. [3] S. Chung, M.R. Sperling, V. Biton, G. Krauss, D. Hebert, G.D. Rudd, P. Doty, Lacosamide as adjunctive therapy for partial-onset seizures: a randomized controlled trial, Epilepsia 51 (2010) 958–967. [4] ICH Q1A (R2), International Conference on Harmonisation, Stability Testing of New Drug Substances and Products, IFPMA, Geneva, 2003. [5] S. Singh, T. Handa, M. Narayanam, A. Sahu, M. Junwal, R.P. Shah, A critical review on the use of modern sophisticated hyphenated tools in the characterization of impurities and degradation products, J. Pharm. Biomed. Anal. 69 (2012) 148–173. [6] R.N. Rao, K. Ramakrishna, B. Sravan, K. Santhakumar, RP-HPLC separation and ESI-MS, 1 H, and 13 C NMR characterization of forced degradants including process related impurities of carisbamate: Method development and validation, J. Pharm. Biomed. Anal. 77 (2013) 49–54. [7] R.N. Rao, B. Ramachandra, K. Santhakumar, RP-HPLC separation and characterization of unknown impurities of a novel HIV-protease inhibitor Darunavir by ESI-MS and 2D NMR spectroscopy, J. Pharm. Biomed. Anal. 75 (2013) 186–191. [8] R.N. Rao, R.M. Vali, S.S. Raju, Liquid chromatography tandem mass spectrometric studies of indinavir sulphate and its forced degradation products, J. Pharm. Biomed. Anal. 74 (2013) 101–110. [9] S.J. Kim, T.S. Koo, D.J. Ha, M. Baek, S.K. Lee, D.S. Shin, H. Moon, Liquid chromatography–tandem mass spectrometry for quantification of lacosamide, an antiepileptic drug, in rat plasma and its application to pharmacokinetic study, Biomed. Chromatogr. 26 (2012) 371–376. [10] C. Kestelyn, M. Lastelle, N. Higuet, S. Dell’Aiera, L. Staelens, P. Boulanger, H. Boekens, S. Smith, A simple HPLC–UV method for the determination of lacosamide in human plasma, Bioanalysis 3 (2011) 2515–2522. [11] V. Sreenivasulu, D.R. Rao, B.N.U. Maheswari, S.K. Das, A. Krishnaiah, Development and validation of a stability-indicating RP-HPLC method for determination of lacosamide, Res. J. Pharm. Biol. Chem. Sci. 2 (2011) 1–11. [12] V.K. Chakravarthy, D.G. Sankar, HPLC method for determination of lacosamide S(−) enantiomer in bulk and pharmaceutical formulation, Rasayan J. Chem. 5 (2012) 293–310. [13] U.K. Chhalotiya, K.K. Bhatt, D.A. Shah, S.L. Baldania, J.R. Patel, Stabilityindicating liquid chromatographic method for quantification of new antiepileptic drug lacosamide in bulk and pharmaceutical formulation, Chem. Ind. Chem. Eng. Q. 18 (2012) 35–42. [14] R.N. Tiwari, C.G. Bonde, Identification and characterization of degradation products of lacosamide by liquid-chromatography/time-of-flight mass spectrometric and multi-stage mass spectrometric analysis, J. Liq. Chromatogr. Relat. Technol. (2014), http://dx.doi.org/10.1080/10826076.2013.825860. [15] ICH Q2 (R1), International Conference on Harmonization, Validation of Analytical Procedures: Test and Methodology, 2005.

MS: development and validation of a stability indicating RP-HPLC method.

The current study dealt with the degradation behavior of lacosamide (LAC) under ICH prescribed stress conditions. LAC was found to be labile under aci...
1MB Sizes 10 Downloads 3 Views