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Research article

Received 29 June 2011, Accepted 5 September 2011 Published online in Wiley Online Library: 12 October 2011

( DOI 10.1002/bmc.1721

Identification and characterization of stressed degradation products of metoprolol using LC/Q-TOF-ESI-MS/MS and MSn experiments
Roshan M. Borkara,b, B. Rajua, R. Srinivasa,b*, Prashant Patelb and Satheesh Kumar Shettyc
ABSTRACT: A rapid, specific and reliable isocratic high-performance liquid chromatography combined with quadrupole time-offlight electrospray ionization tandem mass spectrometry (LC/Q-TOF-ESI-MS/MS) method has been developed and validated for the identification and characterization of stressed degradation products of metoprolol. Metoprolol, an anti-hypertensive drug, was subjected to hydrolysis (acidic, alkaline and neutral), oxidation, photolysis and thermal stress, as per ICH-specified conditions. The drug showed extensive degradation under oxidative and hydrolysis (acid and base) stress conditions. However, it was stable to thermal, neutral and photolysis stress conditions. A total of 14 degradation products were observed and the chromatographic separation of the drug and its degradation products was achieved on a C18 column (4.6 Â 250 mm, 5 mm). To characterize degradation products, initially the mass spectral fragmentation pathway of the drug was established with the help of MS/MS, MSn and accurate mass measurements. Similarly, fragmentation pattern and accurate masses of the degradation products were established by subjecting them to LC-MS/QTOF analysis. Structure elucidation of degradation products was achieved by comparing their fragmentation pattern with that of the drug. The degradation products DP2 (m/z 153) and DP14 (m/z 236) were matched with impurity B, listed in European Pharmacopoeia and British Pharmacopoeia, and impurity I, respectively. The LC-MS method was validated with respect to specificity, linearity, accuracy and precision. Copyright © 2011 John Wiley & Sons, Ltd. Keywords: metoprolol; LC-ESI-MS/MS; degradation products; accurate mass measurements

Metoprolol belongs to the class of selective b1 blocker receptors used in the treatment of several cardiovascular diseases, especially hypertension. It has little or no effect on b2 blocker receptors except in high doses. Treatment of heart failure by b-adrenergic blocking agent has been intensely investigated (Swedberg et al., 1979). Chemically, metoprolol (Scheme 1) is 1-(iso-propylamino)-3-[4′ (2-methoxyethyl) phenoxy]-2-propanol. In the literature, many LC and LC-MS methods have been reported for the analysis of drug in biological fluids and in the presence of other drugs (Balmér et al., 1987; Albers et al., 2005; Yilmaz et al., 2010; Baranowska and Wilczek, 2009). Although Jasińska et al. (2009) carried out stability studies on expired tablets, the study was limited to identification, characterization and the degradation pathway of the drug. The drug substance monograph on metoprolol in the European Pharmacopoeia (2005) and British Pharmacopoeia (2009) lists nine impurities (A–H and J). Of these, four are also mentioned as related substances in the drug monograph in the United States Pharmacopeia (2009). Impurity I is mentioned on the TLC pharmachem website (http://www.tlcpharmachem. com/tlc_item.php?upc=M-0812&li=&sub=). The main aim of the present study was to investigate the complete degradation behavior of the drug and to characterize the degradation products. This was done by exposing the drug to ICH-recommended stress conditions of hydrolysis, oxidation,

thermal and photolysis. The resultant solutions were subjected to optimized LC-MS, MS/MS, MSn and accurate mass measurements to establish the fragmentation pattern of the drug and its degradation products.

Drug and reagents
Pure metoprolol succinate was procured from USP India (P) limited, Hyderabad, India. HPLC-grade methanol and acetonitrile used in the

* Correspondence to: R. Srinivas, National Centre for Mass Spectrometry, Indian Institute of Chemical Technology, Hyderabad, 500 607, India. E-mail:

National Centre for Mass Spectrometry, Indian Institute of Chemical Technology, Hyderabad, 500 607, India National Institute of Pharmaceutical Education and Research, Balanagar, Hyderabad, 500 037, India United States Pharmacopeia–India Private Limited, Research and Development Laboratory, ICICI Knowledge Park, Turkapally, Shameerpet, Hyderabad, 500 078, India Abbreviations used: CID, collision induced dissociation; TOF, time-of-flight.




Biomed. Chromatogr. 2012; 26: 720–736

Copyright © 2011 John Wiley & Sons, Ltd.

Milford. and was used to prepare all solutions Apparatus and equipment Degradation studies were carried out in water bath equipped with a temperature controller. hydrochloric acid and hydrogen peroxide were obtained from Merck (Darmstadt. no. Proposed fragmentation mechanism for metoprolol drug (m/z 268). (Mumbai. MK-10-PH. A photostability chamber (Mack equipment. India). A controlled temperature oven (Mack Pharmatech Private Ltd. 230 V Phase) was used for the photodegradation study. Progard 2 (Millipore. MA.2 Â 106 lx h 200 W h/m2 100  C Exposure 80  C 80  C 80  C Room temperature Photostability chamber Photostability chamber Oven Duration 24 h 48 h 49 h 24 days 4 days 721 Biomed. formic acid. except for methanol and acetonitrile. 2012. Germany). Sr. Ltd. Ammonium formate. 830 . The photostability Table 1. 26: 720–736 Copyright © 2011 John Wiley & Sons. wileyonlinelibrary. present study were purchased from Merck.Degradation products of metoprolol OCH3 OH O O NH m/z 72 -C3H8 O H N m/z 116 -H20 H N m/z 98 m/z 74 -H20 O NH2 m/z 56 H m/z 218 -C3H6 H O m/z 176 -C3H3NH2 H NH2 H3CO H OH H H N m/z 116 -C3H6 O OH NH2 H H3CO -CH3OH H H N m/z 250 H H H3CO m/z 226 -CH3-CH=CH 2 O O OH H3CO -H2O m/z 268 NH2 H C CH H m/z 57 -C12H21NO2 OH H N H H H N -C3H7NH2 H O m/z 191 -CH3OH O m/z 159 -C2H2 O H H OH m/z 121 m/z 133 Scheme 1. USA). All reagents used were at least of analytical grade. HPLC-grade water was obtained by passage through a Milli-QW system. Optimized Stress conditions Stress condition Hydrolysis Acid Base Neutral Oxidation Photolysis Fluorescent light Ultra-violet light Thermal 2 M HCl 1 M NaOH H2O 15 % H2O2 1. Chromatogr. 46/07-08) was used for solid-state thermal stress studies. sodium hydroxide.

Ltd. capillary temperature.85 Æ 0. equipped with an electrospray ionization source.d. Copyright © 2011 John Wiley & Sons. an autoinjector. The other conditions were. Borkar et al. RSD (%) 29.45 mm Chrom Tech Nylon-66 filter and degassed prior to use. The analysis was performed on an Agilent 1200 series HPLC instrument (Agilent Technologies.6 mm i.01 Æ 0. 5 kV. and nitrogen was used as the drying (300  C. The oxidative degradation study was carried out with 15% H2O2 at room temperature for 25 days at a concentration of 1 mg/mL. For collision induced dissociation (CID) experiments. stressed sample solutions were subjected to analysis by a method involving a Waters symmetry C18 column (250 Â 4. MSn experiments were performed using a quadrupole ion trap mass spectrometer (Thermo Finnigan. Separation studies The main objective of this work was to separate metoprolol and its degradations products.13 49. tube lens offset Table 2. 0.R. 26: 720–736 . Ultra high-purity nitrogen was used as the collision gas. 9 L/min) and nebulizing (45 psi) gas.59 Table 3. Acidic and basic hydrolysis was carried out in 2 M HCl. 22308105 Germany). All the solutions were filtered using 0.92 Æ 0. the capillary was set at 3000–3500 V.02 Æ 0. San Jose. SD. The ESI source conditions for MSn studies were: spray voltage. conducted at 80  C with a drug concentration of 1 mg/mL.3032 3672. and an average of 20–25 scans. an on-line degasser. LC-MS analysis was carried out on an Agilent 1200 series HPLC instrument (Agilent Technologies. The data acquisition and processing were carried out using Mass Hunter workstation software.2 Â 106 lx h of fluorescent light and 200 W h/m2 UV-A light in a photostability chamber (ICH. 0. The samples were separated on a Waters Symmetry C18 column (250 Â 4. All the spectra were recorded under identical experimental conditions. flow-rate 1.0527.86 Æ 0.06 99.0417. Other equipment used included a sonicator and a Sartorius balance (CD 225 D.1 mL/min. Parameters of linear regression equation Parameter Calibration range (ng/mL) Correlation coefficient (r2) Slope Intercept SD of slope SD of intercept Value 10–60 0. USA) equipped with an electrospray ionization source.0345. dry heat and photolytic conditions as per ICH (2003) guidelines. All stressed samples were withdrawn at suitable time intervals and diluted 10 times with mobile phase.08 Table 4. 780 pH meter. 0. Agilent Technologies. M.0374. 0.06 60. a diode-array detector.16 30.90 Æ 0. 200  C. particle size 5 mm).6 mm. detection wave length 225 nm and column temperature 25  C. and changing the organic modifier to acetonitrile. RSD (%) 24. RSD (%) 29. Several studies were carried out by changing ratio of acetonitrile until satisfactory resolution was obtained. SD. A splitter was placed before the electrospray ionization source.0444. chamber consisted of both UV and fluorescent lamps. Germany) with an Epson printer Lx-300 t. respectively. The typical Q-TOF operating source conditions for MS scan of metoprolol in this mode were optimized as follows: the fragmentor voltage was set at 80 V. for 24 and 48 h.22 mm membrane filters before HPLC and LC-MS analysis. whereas neutral hydrolysis was carried out in water for 48 h. Data of intra-day and inter-day precision studies (n = 3) Concentration (ng/mL) 30 50 60 Intra-day precision. even with varying pH. The data acquisition and processing were under the control of Xcalibur software. For thermal stress. 2012.11 Recovery (%) 99. 0.07 60. Stressed degradation studies Stress degradation studies of metoprolol were carried out under hydrolysis (acid.0397. A calibrated lux meter and UV meter were used to measure energy.6 100. base and neutral). measured concentration (ng/mL).. USA).94 Æ 0. good separation was not achieved. 15–20 V. CA. measured concentration (ng/mL). USA).05 Inter-day precision.14 34. USA) coupled to a quadrupole time-of-flight mass spectrometer (Q-TOF LC/MS 6510 series classic G6510A. 0. keeping MS1 static. SD. and the pressure in the collision cell was maintained at 18 Torr. Chromatogr. Recovery data for metoprolol spiked into a mixture of stressed samples Spiked concentration (ng/mL) 25 30 35 Calculated spiked concentration (ng/mL). The optimized stressed conditions are outlined in Table 1.90 Æ 0. Biomed. All the hydrolytic studies were MS/MS and MSn studies of the drug The fragmentation pathway of metoprolol was established by carrying out TOF-MS/MS and MSn studies in positive-ion ESI mode. the drug was kept at 100  C in the oven for 4 days.0394. capillary voltage. Initially. 0.84 722 wileyonlinelibrary.0542. a column oven and a computer system embedded with Chemstation software. 0. 1 M NaOH. allowing entry of only 35% of the eluent. particle size 5 mm) and a mobile phase comprising a mixture of 20 mM ammonium formate (pH adjusted to 3 by formic acid) and methanol. The HPLC system consisted of a quaternary pump. The mobile phase was filtered through a 0. All pH measurement was done using a pH-meter (Metrohm Schweiz AG. 1996). However. the skimmer was 60 V. ratio of mobile phase components and flow-rate.9998 13654 4590 94.18 49. the precursor ion of interest was selected using the quadrupole analyzer and the product ions were analyzed using a time-of-flight (TOF) analyzer.11 Æ 0. oxidation. Solid-state photolytic studies were carried out by exposing light to a thin layer (1 mm) of drug in a Petri dish to 1.0354.

the automatic gain control settings were 2 Â 107 for a full-scan mass spectrum and 2 Â 107 counts for a full-product ion mass spectrum with a maximum ion injection time of 200 ms. (d) LC-ESI-MS-TIC of oxidation degradation products. the precursor ion of interest was first isolated by applying an appropriate waveform across the endcap electrodes of the ion trap to resonantly eject all trapped ions. 30 psi.Degradation products of metoprolol Figure 1. and helium used as damping gas. (b) LC-ESI-MS-TIC of neutral/photolysis/thermal degradation products. 20 V. 26: 720–736 Copyright © 2011 John Wiley & Sons. Chromatogr. . sheath gas (N2) pressure. 2012. For the ion trap mass analyzer. wileyonlinelibrary. In full-scan MS2 and MS3 modes. The isolated ions were then 723 Biomed. (c) LC-ESI-MS-TIC of base degradation products. except those ions of the m/z ratio of interest. Ltd. (a) LC-ESI-MS total ion chromatograms (TIC) of acid degradation products.

8 8.62 À2.22 5.1070 58. 151. 99. 149.0570 222. 111. 98 116. 133.0 18. 133.1910 135.1051 238. 176. 135.1172 153. Drug DP1 DP2 DP3 DP4 DP5 DP6 DP7 DP8 DP9 DP10 DP11 DP12 DP13 DP14 10. 56 — 383.66 À6. 91 226.5 12 15. 116 — 163.1645 À2.0 5.0581 222.1811 226. 2012. 159. 268. 117.12 À3.1067 238. 232. 98. 98. 151. 133.8 4.1161 153. 121. M.0804 250. 72. 159.0651 383. 99.0801 250. 191. 74. 56. 218. 159.2 17. 91. 135.11 — C3H7NH2 C6H13NO C6H15NO2 H2O C6H13NO2 C5H11NO CH2O C3H6 C9H12O2 C12H18O3 IN* C12H18NO2 C2H6O CH4O Calculated mass (Da) Error (ppm) Proposed neutral loss MS/MS fragment ions 250.12 À3. 194. 121. 306.8 C15H26NO3 C12H17O3 C9H13O2 C9H11O C15H24NO2 C9H13O C10H15O2 C14H24NO2 C12H20NO3 C6H14NO C3H8N C21H39N2O4 C3H8O C13H20NO2 C14H22NO2 a Interaction product having m/z 383. 191. 69 — 250.18 À4.6 5.16 4. Chromatogr.2911 60. 105. 57. 58 177. 98.1802 226.724 Observed mass (Da) 268. 158. 165.2 6.2904 60. 105. 148.12 1.1815 137. 149.15 À3. Borkar et al. 72. 59.16 À2.1432 116. 149. 226. 56 R.3 2.1912 209. 74. Ltd.0985 135. 73.12 À2. 59. Elemental compositions for metoprolol and its degradation products Drug/degradation product Rt (min) Proposed formula Copyright © 2011 John Wiley & Sons.91 À4. 123. 176. 26: 720–736 . 72 167.165.9 3.1489 236. 133. 121. 57 153. 74.58 0.1092 58. 218.1438 116.1907 209. 57.8 6. 56.1471 236. 56 — — 179. 116.0655 383.1655 268. Table 5. Biomed.0 18. 324. 105. 74.1802 137.0967 167. 116. 99. 176.11 À2.3 3.0961 167. 74.

(d) LC-ESI-MS/MS spectrum of [M + H]+ ions (m/z 250) of DP4 at 25 eV. (i) LC-ESI-MS/MS spectrum of [M + H]+ ions (m/z 222) of DP13 at 32 eV. . (a) LC-ESI-MS/MS spectrum of [M + H] + ions (m/z 268) of metoprolol at 26 eV. 26: 720–736 Copyright © 2011 John Wiley & Sons. (e) LCESI-MS/MS spectrum of [M + H]+ ions (m/z 238) of DP7 at 32 eV.Degradation products of metoprolol Figure 2. (b) LC-ESI-MS/MS spectrum of [M + H]+ ions (m/z 209) of DP1 at 20 eV. 725 Biomed. (g) LC-ESI-MS/MS spectrum of [M + H]+ ions (m/z 116) of DP9 at 16 eV. (c) LC-ESI-MS/MS spectrum of [M + H] + ions (m/z 153) of DP2 at 16 eV. (f) LC-ESI-MS/MS spectrum of [M + H] + ions (m/z 226) of DP8 at 21 eV. (h) LC-ESI-MS/MS spectrum of [M + H]+ ions (m/z 383) of DP11 at 21 eV. Ltd. Chromatogr. wileyonlinelibrary. (j) LC-ESI-MS/MS spectrum of [M + H]+ ions (m/z 236) of DP14 at 22 eV.

726 wileyonlinelibrary. Figure 2. 26: 720–736 . 2012. Borkar et al. Ltd. (Continued). M. Copyright © 2011 John Wiley & Sons.R. Biomed.

1070 57.0444 57. wileyonlinelibrary.1070 226.0336 98.12 À . 2012. The degradation products were analyzed by MS/MS.1912 250. all the spectra were recorded with an average of 25–30 scans.18 À2.0336 Calculated mass (Da) 268.0335 98. MSn fragmentation and accurate mass measurements.92 1.96 3.1802 191. The excitation time used was 30 ms.1054 159. signal to resonantly excite them and so cause CID.12 2. metoprolol was analyzed on a C18 column (250 Â 4.0611 56.0632 218. Chromatogr.16 0.c.1071 226. Under these conditions the peak shape of the drug was not satisfactory. 727 Biomed.1539 176.6 mm i. Table 6.0954 74.d. particle size 5 mm) using 20 mM ammonium formate buffer (pH 3) and methanol in the ratio of 80:20 at a flow-rate of 1 mL/min.62 2.Degradation products of metoprolol Figure 2.0491 121. Subsequent trials were made on a mixture of degraded samples by changing organic modifier to LC-MS/TOF studies on degradation products Both the drug and degraded samples were investigated using LC-MS/ TOF mass spectrometry. Elemental compositions for daughter ion of metoprolol.0451 57. Ltd.1532 176. 26: 720–736 Copyright © 2011 John Wiley & Sons.0964 74. (Continued).0814 133.91 À4.0648 218.1432 116.0648 72.1067 159.0641 72.16 Proposed neutral loss — H 2O C3H7NH2 CH3OH C2 H 2 CH3OH C3 H 6 CH3—CH&dbond.1092 57. m/z 268 Drug Proposed formula C15H26NO3 C15H24NO2 C12H15O2 C11H11O C9 H 9 O C14H20NO C11H14NO C12H20NO3 C6H14NO C3 H 5 O C6H12N C3H8NO C3 H 6 N C8 H 9 O C3H6NO C3 H 5 O Observed mass (Da) 268.CH2 C9H12O2 C12H21NO H 2O C3 H 6 H 2O C3H3NH2 C3 H 8 C12H21NO Metoprolol subjected to a supplementary a.1907 250. The collision energies used were 20–35 eV. Results and discussion Development and optimization of LC and LC-MS method Initially.1815 191.62 À1.16 2..15 À0.98 À2.0804 133.1438 116.12 À0.0335 Error (ppm) À2.21 À4.0495 121.98 0.0600 56.

Ltd.R. Borkar et al. O O CH 3 H m/z 73 H O O HC O CH3 m/z 99 H3CO OCH3 CH2 m/z 59 H3CO H OCH 3 HC m/z 57 H2C m/z 167 -CH3OH O CH 3 m/z 135 -CH2O H H2 C m/z 105 H -C7H8O m/z 209 -C2H2O O CH3 m/z 121 H H -C7H10O -C9H12O O O CH3 H -CH 3-CH2 OCH 3 O CH3 m/z 149 .CO O CH 3 -C6H4O m/z 117 -C7H10O Scheme 3. M. 728 Copyright © 2011 John Wiley & Sons. O O CH3 H3CO DP1 m/z 209 H OH H H H3CO DP2 m/z 153 H3CO DP3 m/z 135 O H N H H O CH3 H H3CO DP4 m/z 250 H3CO DP5 m/z 137 H3CO DP6 OH O NH2 H CH3 O m/z 167 H H N m/z 116 OH O H N H m/z 238 DP7 H HN DP10 m/z 58 H3CO DP8 m/z 226 OH O H N CH2 H N DP9 H H2N DP12 m/z 60 H3CO m/z 383 DP11 OH O H N H O HO OH NH H H3C DP13 m/z 222 DP14 m/z 236 Scheme 2. Chromatogr. 2012. Proposed fragmentation mechanism for DP1 (m/z 209). Proposed structures of degradation products formed under various stress conditions. Biomed. 26: 720–736 .

0335 250.0441 73.0648 117.0491 57.1 mL/min and detection wave length was maintained at 225 nm.68 5.12 0. a stock solution containing 1 mg/mL metoprolol in mobile phase was diluted to yield solutions in the concentration range of 10–60 ng/mL.0712 Calculated mass (Da) 209. 2012. wileyonlinelibrary.1234 74.0699 99.14 4.1071 98.0812 56.0491 218. without replacement of buffer. precision (inter-day.1539 176.1226 74. spray voltage and skimmer voltage were optimized to maximize the ionization in the source and sensitivity even at a very low concentration to identify and characterize the degradation products.0440 73. accuracy and .0597 135.0582 135.1061 149. pH 3): acetonitrile–methanol in the ratio of 85:15:5.0804 121. The solutions were prepared and analyzed in triplicate.62 À2.0681 99. Proposed fragmentation mechanism for DP2 (m/z 153). Elemental compositions for daughter ions of DP1 (m/z 209).11 4.62 Proposed neutral loss — C2H2O CH3—CH2—OCH3 CH3OH CO C6H4O CH2O C7H10O C9H12O C7H8O C7H10O — C2H2O C5H8O — C3H6O C3H7 C6H5O CH3OH C3H6 C9H12O2 C5H8O C11H12O2 DP2 DP4 H OH H3CO m/z 153 -C5H8O -C2H2O H OH H H3CO m/z 69 m/z 111 Scheme 4.16 À6. The Table 7. For LC-MS studies.92 0.0336 153.1161 167.66 2.91 À3. Ltd. The Q-TOF ESI source conditions were also optimized to obtain a good signal and high sensitivity.23 À1. The linearity data are given in Table 2.0964 Error (ppm) À4.0911 105.12 À4.15%.0284 59.0812 69.11 4.62 À3.1802 191. The best separation with good peak shape was achieved on the same column at 25  C using a mobile phase composed of an ammonium formate buffer (20 mM.0910 105. nebulizing gas flow.10 0.Degradation products of metoprolol acetonitrile.0835 56. 729 Biomed.1078 149.1910 111.1067 149.1815 191.0804 69.22 À0.0985 111. same method was used as for HPLC.92 1.0335 153.11 4. drying gas temperature. intra-day and intermediate precision).0801 121.26 À0.11 5. capillary voltage.1536 176. The flow-rate was 1. To establish linearity and range. DP2 (m/z 153) and DP4 (m/z 250) Degradation product DP1 Proposed formula C12H17O3 C10H15O2 C9H9O2 C9H11O C8H9O C6H13O2 C8H9 C5H7O2 C3H5O2 C3H7O C3H5O C9H13O2 C7H11O C4H5O C15H24NO2 C12H17NO C9H11NO C3H6N C14H20NO C11H14NO C6H12N C10H16NO C4H12N Observed mass (Da) 209.0336 250.1305 149.12 À2. Chromatogr.0495 218.0482 57.0652 117.11 À0.1070 98. 26: 720–736 Copyright © 2011 John Wiley & Sons. Method validation The stability-indicating method was validated for linearity. The optimized LC-MS method was validated with respect to various parameters summarized in the ICH (2005) guidelines. The conditions like drying gas flow.0954 166.0964 166. The response for the drug was linear in the investigated concentration (r2 = 0.0266 59.1172 167.98 2.9998) and the %RSD for each investigated concentration was <0.

resulting in eight degradation products (DP1. Proposed fragmentation mechanism for DP4 (m/z 250). DP5 and DP9. DP1-DP14 (Table 5). m/z 116 (loss of 4-(2-methoxyethyl)phenol). 1a) and after 4 days seven degradation products (DP3.and inter-day precisions were determined at three different concentrations. intra. DP13 and DP14) were formed. Borkar et al. m/z 121 (protonated 4-vinylphenol). on the same day (n = 3) and consecutive days (n = 3).1(b). extensive degradation was observed in 15% H2O2 after 25 days (Fig. 1c). The degradation products are given different notations. In neutral conditions. The proposed structures and elemental compositions of all the degradation products are shown in Scheme 2 and Table 5. MS/MS CID of degradation Copyright © 2011 John Wiley & Sons. as shown in Fig. respectively. DP3. 2a. Johnson and Lewis. and was also demonstrated by subjecting all the degradation samples to LC-MS. Oxidative stress. m/z 57 (protonated methoxyethyne) and m/z 56 (protonated propa-1. The drug was stable to light and thermal stress in the solid state. The MS/MS spectrum (Fig. The LC-ESI-MS total ion chromatograms obtained under various stress conditions are given in Fig. were observed on treatment of the drug in 1 M NaOH for 48 h at 80  C (Fig. DP7. To characterize all the degradation products formed under various stress conditions. instead. m/z 226 (loss of propene). M. Photolysis and solid-state studies. DP8. no degradation product was formed (Fig. m/z 159 (loss of methanol from m/z 191). Table 3 shows that the %RSD for intraand inter-day precision was <0. m/z 74 (protonated 3-aminoprop-1-en-2-ol). Degradation behavior The optimized LC-MS method is applicable for identifying the degradation products. indicating that the method was sufficiently precise. DP10. which unambiguously proves the specificity of the method. two degradation products. 2012. m/z 98 (loss of H2O from m/z 116). The positiveion ESI mass spectrum of metoprolol shows an abundant [M + H]+ ion at m/z 268. 1. 50 and 60 ng/mL. viz. m/z 176 (loss of propene from m/z 218). m/z 218 (loss of methanol from m/z 250). indicating that the drug is stable. Table 4 shows that recoveries of the added drug were obtained from the difference between peak areas of fortified and unfortified degraded samples. 1b). 26: 720–736 . LC-MS/MS and MSn studies of metoprolol and its degradation products Fragmentation pathway of the protonated drug. 2006) shows product ions at m/z 250 (loss of H2O). The drug showed an extensive degradation in 2 M HCl at 80  C (Fig. Biomed. Chromatogr.20% respectively. 1d). All these fragmentation pathways were confirmed by MSn experiments and accurate mass measurements (Table 6). 730 wileyonlinelibrary. In comparison to neutral stress conditions. m/z 191 (loss of propan-2-amine from m/z 250).R. Hydrolysis. DP2. DP6. Scheme 1). DP9 and DP11). A total of 14 degradation products were identified and characterized by tandem mass spectrometric analysis (LC-ESI-MS/MS). No significant degradation was observed in 3% H2O2 after 48 h at room temperature. 30. H H N H3C m/z 98 O m/z 166 H H N H N H H N H3C m/z 74 H3CO -CH3OH H N H O -C5H8O m/z 250 -C3H7O H N O m/z 218 -C3H6 O m/z 191 -C3H6 H O m/z 149 -C6H5O NH 2 m/z 56 NH 2 O NH2 m/z 176 Scheme 5. The specificity of the method was established by determining peak purity for metoprolol in a mixture of stressed samples using a photodiode array (PDA) detector and evaluation of the resolution factor.2-dien-1-amine. DP4. Ltd. DP12. The mass detector showed an excellent purity for metoprolol and every degradation product. on heating the drug in water for 4 days at 80  C. the CID of the protonated degradation products was carried out using Q-TOF and ion trap mass spectrometers. m/z 72 (protonated 1-iminopropan-2-one). DP6.15 and <0. m/z 133 (loss of C2H2 from m/z 159).

Scheme 5) and low-abundance ions at m/z 218 (loss of methanol). The formation of m/z 98 is indicative of the presence of the N-isopropylprop-1-en-1-amine group. 731 Biomed. Fig. m/z 105 (protonated styrene). m/z 121 (loss of CO m/z 149). 2b. m/z 250). 26: 720–736 Copyright © 2011 John Wiley & Sons. m/z 117 (loss of phenol). m/z 153). The ESI-MS/MS spectrum of [M + H]+ ions (m/z 250) of DP4 (Rt = 4. m/z 149 (loss of CH3–CH2–OCH3). m/z 176 and m/z 149 points to the presence of phenoxy-N-isopropylprop-1-en-1-amine moiety in the structure of DP4. m/z 191 (loss of C3H7O). The DP2 formed under oxidative stress conditions eluted at Rt 3. was 18 Da less than the drug. Chromatogr. The [M + H]+ ion of DP2 gave abundant product ions at m/z 111 (loss of C2H2O from m/z 153) and m/z 69 (loss of C5H8O from m/z 153. The formation of fragment ions at m/z 218. wileyonlinelibrary. The molecular mass of DP2 was found to correspond to the molecular mass of impurity B (European and British pharmacopoeias). m/z 57 (protonated methoxyethyne) and m/z 59 ( . m/z 99 [protonated 1-(ethynyloxy)propan-2-one]. The elemental compositions of all fragment ions were confirmed by accurate mass measurements (Table 7). All these data are consistent with the proposed structure 1-[4-(2methoxyethyl) phenoxy] propan-2-one proposed for DP1. m/z 176 (loss of C3H6 from m/z 218). Proposed fragmentation mechanism for DP7 (m/z 238). These characteristic fragment ions were highly compatible with the structure of 4-(2-methoxyethyl) phenol. 2012. m/z 73 (protonated 2-oxopropanal).9 min). m/z 149 (loss of propene from m/z 191). m/z 191. The ESI-MS/MS spectrum of [M + H]+ ions (m/z 209) of DP1 (Rt = 2. m/z 74 (protonated N-methylpropan-2amine).3 min.6 min). These fragmentation pathways have been confirmed by MSn experiments and accurate mass measurements (Table 7). The CID spectrum showed abundant product ions at m/ z 166 (loss of C5H8O) and m/z 98 (loss of C9H12O2. DP4 ([M + H]+. shows product ions at m/z 167 (loss of C2H2O). Fig. m/z 209).Degradation products of metoprolol DP1 ([M + H]+. 2c. suggesting a loss of water molecules from the protonated drug (m/z 268). All these fragmentation pathways were confirmed by MSn experiments. 2d. Fig. Scheme 3). Scheme 4). m/z 135 (loss of methanol from m/z 167). Ltd. The elemental compositions of protonated DP4 H O H2C m/z 159 -C3H13NO -C6H15NO2 C H3C m/z 105 CH2 H NH m/z 72 OH O H3C m/z 238 NH -C3H7NH 2 OH O H3C m/z 179 H CH2 m/z 91 O O HC O m/z 99 H CH3 H CH3 -C6H8 O H3C m/z 179 -C2H4 O O m/z 151 -CO -H20 H CH3 O CH3 H CH2 O CH3 O m/z 133 H CH m/z 123 m/z 57 Scheme 6. m/z 56 (C3H6N+). The formation of fragment ions at m/z 57 and m/z 59 is indicative of the presence of methoxyethyl group while fragment ions at m/z 121 and m/z 149 authenticate the presence of phenoxy moiety in DP1. which may be attached to phenoxy moiety. DP2 ([M + H]+.

0631 56.0335 133. The DP9 formed under both oxidative and basic stress conditions eluted at an Rt of 8. and its fragment ions were confirmed by accurate mass measurements (Table 7). DP9 ([M + H]+. The elemental compositions of DP8 and its fragment ions were confirmed by accurate mass measurements (Table 8). DP7 ([M + H]+. Scheme 7). m/z 133 (loss of H2O from m/z 151). m/z 268 (protonated metoprolol) and m/z 116 [protonated 1-(isopropyl amino) propan-2-one]. m/z 159 (loss of methanol from m/z 191).0706 98.1172 165. m/z 105 (1-ethylbenzene cation).2 min) was 30 Da less than the protonated drug.0910 176. These + + fragmentation pathways were confirmed by MSn experiments. as shown in Scheme 9. the fragment ions at m/z 179.0632 74.0808 91.8 min) displayed abundant product ions at m/z 121 (4-vinylphenol) and m/z 74 (1-aminopropan-2one.58 0.0814 194. m/z 123 (loss of CO from m/z 151). respectively.0495 Error (ppm) À2.2-dien-1-amine). Fig.0611 121. 2012.0804 105.0814 123. m/z 165 (loss of methanimine from m/z 194). 26: 720–736 .0600 121.0759 57.0336 133.0648 72. Based on these data.16 0.1432 159.1065 99.92 À5. which eluted at an Rt of 15. m/z 99 [protonated 1-(ethynyloxy) propan-2-one.(isopropyl amino) propan2-ol proposed for DP7. The ESI-MS/MS spectrum of [M + H]+ ions (m/z 238) of DP7 (Rt = 6.0812 105. Chromatogr. 2g) The elemental compositions of DP9 and its fragment ions were been confirmed by accurate mass measurements (Table 9).0757 116. M. m/z 306 (loss of H2O from m/z 324).1067 99. As can be seen from Scheme 6. m/z 123 and m/z 105 are highly compatiable with the structure 1-(4-ethylphenoxy)-3.1070 133. m/z 116).24 À1.0804 194. and the fragment ions at m/z 98.10 À4.96 À3.0698 226.14 4.0648 148.0511 159. m/z 98 [protonated 3-(ethynyloxy) prop-1-en-1amine] and m/z 56 (protonated propa-1.(ethynyloxy) propan-2-ol). The MS/MS. m/z 151 (loss of C2H4 from m/z 179 ). m/z 72 (N-methylpropan-2-amine cation) and m/z 91 (C7H+ 7 ).0672 148.92 Proposed neutral loss — C3H7NH2 C6 H 8 C2 H 4 C6 H 6 O H 2O C10H14O2 C7H17NO2 C3H13NO CO C6H15NO2 — NH3 CH4O CHNH2 H 2O C2 H 2 C8 H 8 O C3H5ONH2 C2 H 4 C7H10O H 2O H 2O DP8 732 wileyonlinelibrary. The CID of protonated DP7 yielded abundant product ions at m/z 179 (loss of C3H7NH2). m/z 226).0491 Calculated mass (Da) 238. DP11 (m/z 383).1802 179.0929 176.0648 74.16 2. m/z 159. Ltd.0804 123. MSn Table 8. m/z 238). Elemental compositions for daughter ions of DP7 (m/z 238) and DP8 (m/z 226) Degradation product DP7 Proposed Formula C14H24NO2 C11H15O2 C5H7O2 C9H11O2 C3H5O C9H9O C4H10N C7H7 C11H11O C8H11O C8H9 C12H20NO3 C11H12O C11H16NO2 C10H13O2 C11H14NO C9H9O C3H8NO C8H9O C9H10NO C5H10NO2 C5H10NO2 C9H11O2 Observed mass (Da) 238.0711 98.0445 151. m/z 57 (propan-2-one cation). DP8 ([M + H] . m/z 57 (C3H5O+) and m/z 59 (protonated propan-2-one.1071 133.98 2.1176 165. m/z 176 (loss of H2O from m/z 194). 2f.0441 151.89 À0.16 0.12 À3.0600 56.96 À1. It can be noted that DP9 corresponds to the complementary product ion of DP2 (Scheme 8). and low-abundance ions at m/z 194 (loss of methanol).0632 72. DP9 was identified as protonated 1-(isopropyl amino) propan-2-one. Borkar et al.0768 116. The characteristic fragment ions at m/z 116 and 121 are indicative of the presence of 1-aminopropan-2-ol and phenoxy groups in DP8. and 54 are diagnostic for the presence of 3-(ethynyloxy) prop-1-en-1amine moiety.32 0.16 À4. m/z 148 (loss of C2H4 from m/z 176). The ESI-MS/MS spectrum of [M + H] ions (m/z 226) of DP8 (Rt = 6. The formation of fragment ions at m/z 268 and m/z 116 indicates that DP11 can be formed by the combination of corresponding moieties. m/z 116 (protonated 1-amino-3. Figure 2(h) shows the ESI-MS2 spectrum of [M + H]+ ions (m/z 383) of DP11.16 À5. These fragmentation pathways were confirmed by MSn experiments and accurate mass measurements.91 À2.R. Biomed. suggesting a neutral loss of formaldehyde from the protonated drug (m/z 268).0754 57. 76.0699 226. All these data are highly compatible and confirm the structure 1-[4-(2-methoxyethyl) phenoxy]-3-aminopropan-2-ol proposed for DP8. All these fragmentation pathways were confirmed by MSn experiments and accurate mass measurements (Table 8). Scheme 6].22 À2.1438 159.0542 159.2 min. The protonated DP9 yielded product ions at m/z 74 (loss of propene). and low-abundance ions at m/z 159 (loss of C3H13NO). 2e. m/z 56 (loss of H2O from m/z 74). Fig.16 À0. The protonated DP11 yielded products ions at m/z 324 (loss of C3H7NH2).12 1. m/z 332 (loss of C6H5O from m/z 324). m/z 133 (loss of C2H2 from m/z 159).0811 91.98 Copyright © 2011 John Wiley & Sons. Fig.1811 179.98 À3. m/z 133.5 min. All these data are consistent with the proposed structure 3-[4-(2-methoxyethyl) phenoxy]-N-isopropylprop-1-en-1amine for DP4.

m/z 135) was identified as protonated 1-(2-methoxyvinyl) benzene. DP3 ([M + H]+. m/z 99 (loss of C5H4 from m/z 163) and m/z 58 (propan-2-imine cation.2i. Fig. 733 Biomed. respectively. m/z 133 (loss of C2H2 from m/z 159). wileyonlinelibrary. m/z 236). the possible structure 1-(4-vinylphenoxy)-3-(isopropyl amino) propan-2-ol can be assigned to DP14. DP5 and DP6 were identified as protonated 1-(2-methoxyethyl) benzene and protonated 1-methoxy-4-(2 methoxyethyl) benzene. whereas DP6 formed under both acidic and oxidative stress conditions (Rt = 5. Proposed fragmentation mechanism for DP8 (m/z 226).0 and 5. Based on the fragmentation pattern and accurate mass measurements. The molecular mass of DP14 was found to correspond to the molecular mass of impurity I (www. fragment ions and elemental compositions derived from accurate mass measurements (Table 9) are consistent with the proposed structure 1-({2-hydroxy-3-[4-(2-methoxyethyl) phenoxy] propyl} (isopropyl) amino)-3-(isopropyl amino) propan2-ol for DP11. m/z 222). respectively). DP3 (Rt 3.2-dien-1amine). m/z 137) and DP6 ([M + H]+. DP13 ([M + H]+. m/z 149 (loss of C4H9NH2).0 min. The DP13 formed under acidic stress conditions eluted at the Rt of 18. m/z 72 (C4H10N+) and m/z 56 (C3H6N+. Similarly. All these data are in line with the structure 1-(p-tolyloxy)-3-(isopropyl amino) propan2-ol proposed for DP13.Degradation products of metoprolol OH H NH2 m/z 56 -H2O H O NH2 m/z 74 -C8H8O -CH3OH OH H OH -C3H5ONH2 O H3CO m/z 226 O m/z 116 -C7H10O O NH2 H H -H2O O m/z 98 NH2 OH NH2 H -H20 H O NH2 H3CO m/z 208 -NH3 H NH2 H3CO O H m/z 191 m/z 194 m/z 121 CH2=NH -H2O O OH O H m/z 176 -NH3 m/z 165 -C2H2 H O H O NH2 m/z 159 m/z 150 -C2H2 H O m/z 148 O NH2 -C2H4 NH2 H m/z 133 Scheme 7.8 min) displayed abundant product ions at m/z 98 (protonated N-isopropylpropa-1. The mass difference between DP5 and DP6 was CH2O (30 Da). DP5 ([M + H]+. The DP5 formed under basic stress conditions. m/z 105 (protonated styrene). DP14 ([M + H]+. m/z 135). m/z 167). Scheme 11). Ltd. 2012.tlcpharmachem. m/z 177 (loss of propan-2-amine) and low-abundance ions at m/z 159 (loss of H2O from m/z 177). Fig. m/z 151 (loss of C2H2 from m/z 177). m/z 91 (C7H7+). m/z 135 (loss of CO from m/z 163).8. Scheme 10). m/z 74 (protonated N-methylpropan2-amine). 26: 720–736 Copyright © 2011 John Wiley & Sons.8 min. The ESI-MS/MS spectrum of [M + H]+ ions (m/z 236) of DP14 (Rt = 18. The ESI-MS of DP5 and DP6 showed abundant [M + H]+ ions at m/z 137 and m/z Based on elemental compositions and accurate mass measurements (Table 5). These fragmentation pathways were confirmed by MSn experiments. The ESI-MS2 spectrum of [M + H]+ ions (m/z 222) of DP13 displayed product ions at m/z 163 (loss of C3H7NH2). The elemental compositions of protonated DP13 and its fragment ions were confirmed by accurate mass measurements (Table 9).com/journal/bmc . The elemental compositions of DP14 and its fragments ions were confirmed by accurate mass measurements (Table 9). Chromatogr. 2j. respectively.

0648 105.0812 99. DP10 ([M + H]+.91 3.0808 56.0964 91.89 2.1912 116.62 À2.61 4. 26: 720–736 . Chromatogr.26 1.0412 74.0335 383.0632 105.1489 163.0811 56. Proposed fragmentation mechanism for DP9 (m/z 116).com/journal/bmc Copyright © 2011 John Wiley & Sons. 734 wileyonlinelibrary.1092 74.1655 177.2064 232. respectively.96 4.12 5.11 À1.0495 Error (ppm) À4.0336 383.0814 151.0600 59.0481 56. DP11 (m/z 383).0804 99.0491 Calculated mass (Da) 116.0597 135.0911 159.0964 58.1907 116.2171 306.0681 98. Borkar et al.14 0. m/z 60).0495 57.0651 236.0441 74.0 and 17.1907 268.91 À3.1645 177.2053 232. Table 9. Ltd.R.0699 98.0655 236.16 À4.0754 149.0759 133. DP13 (m/z 222) and DP14 (m/z 236) Degradation product DP9 Proposed formula C6H14NO C3H8NO C3H9N C9H11O2 C3H5O C21H39N2O4 C18H30NO4 C18H28NO3 C12H26NO3 C15H26NO3 C6H14NO C13H20NO2 C10H11O2 C9H9O2 C9H11O C5H7O2 C4H12N C3H8N C14H22NO2 C11H13O2 C11H11O C9H11O2 C9H9O C8H9 C6H12N C7H7 C4H10N C3H6N Observed mass (Da) 116.0611 59. respectively. Biomed.0912 58.0954 91.62 4.92 À0.12 À1.2904 324.12 1.0910 159.0542 72.1070 222.10 2.0591 135. m/z 58) and DP12 ([M + H]+. The DP10 and DP12 formed under acidic stress conditions with Rt of 12.2911 324.12 5.18 À4.0804 151.1092 222.66 0.0491 56.0511 72.1911 268.96 À2. Elemental compositions for daughter ions of DP9 (m/z 116).22 À3.12 À1.0 min.0751 149.16 À3. Based on elemental compositions and accurate mass measurements (Table 5).15 4.2169 306.12 À3.1070 74.0754 133.1471 163. DP10 and DP12 were identified as protonated propan-2-imine and protonated propan-2-amine.92 Proposed neutral loss — C3H6 C3H5NH2 H2O NH3 — C3H7NH2 H2O C6H5O C6H13NO C9H12O2 — C3H7NH2 C4H9NH2 CO C5H4 C9H8O2 C1OH12O2 CH4O C3H9N H2O C2H2 C2H2 C6H13NO2 C8H10O2 C7H15NO2 C10H12O2 C11H16O2 DP11 DP13 DP14 OH NH H 2C H O NH H3 C m/z 116 -C 3H6 -C3H5NH2 O H H3C CH3 O H3C m/z 74 -NH 3 OH H2C H 3C m/z 57 CH m/z 56 NH 2 H m/z 59 -H2O NH2 H Scheme 8.16 3.0491 57. 2012.16 À2. M.

respectively. Conclusions Stress degradation studies on metoprolol. Proposed fragmentation mechanism for DP11 (m/z 383). wileyonlinelibrary. . provided information on the degradation behavior of the metoprolol under the conditions of hydrolysis and oxidation.Degradation products of metoprolol OH O H N CH2 H3CO m/z 383 -C6H13NO OH O H2 N H3CO O -C3H7NH2 OH H N CH2 m/z 324 -C6H4O OH H N m/z 116 H3CO H N CH2 m/z 232 HO m/z 306 HO -H2O HO H N H3CO m/z 268 -C9H12O2 OH Scheme 9. 735 Biomed. carried out according to ICH guidelines. Proposed fragmentation mechanism for DP13 (m/z 222). A total of 14 degradation products were characterized with the help of the MS/MS. thermal and photolytic stress conditions. The drug showed extensive degradation in acid. MSn experiments combined with accurate mass measurements of fragment ions and precursors. 2012. base hydrolysis and oxidative stress. The liquid chromatography method described in the present study can resolve all the degradation products from the metoprolol as well as from each other under various stress conditions. 26: 720–736 Copyright © 2011 John Wiley & Sons. DP2 (m/z 153) and DP14 (m/z 236) matched impurities B and I. H NH H3C m/z 74 -C9H8O2 OH O H2N m/z 58 CH 3 -C3H7NH 2 OH O CH2 CH3 m/z 163 -CO -C5H4 OH O CH2 H3C m/z 135 CH m/z 99 m/z 222 NH H -C4H9NH 2 CH3 m/z 149 O OH H O CH 2 Scheme 10. Of these degradation products. Ltd. while it was stable to neutral.

International Conference on Harmonization. 1996. alpha. IICT. International Conference on Harmonization. Völker C. Copyright © 2011 John Wiley & Sons. 2009. Biomed. Analytical Science 2009. United States Pharmacopeia. Hyderabad for facilities and their cooperation. Asci A and Arslan S. NIPER. 5thEdition. Baranowska I and Wilczek A.R. is thankful to DST for the award of a Junior Research Fellowship. 3. HPLC quantification of metoprolol with solid-phase extraction for the drug monitoring of pediatric patients. 25(6): 769–772. Johnson RD and Lewis RJ. medicinal and pharmaceutical substances. Stability Testing of New Drug Substances and Products. 1: 1374–1376. B. 2005. ICH. 5: 2032–2033. I and II 3933–3936.R. 2012. 2963–2970. Acta Poloniae Pharmaceutica 2009. Determination of metoprolol and two major metabolites in plasma and urine by column liquid chromatography and fluorometric detection. 26: 720–736 . paracetamol and its glucuronide and sulfate metabolite in human urine. Yilmaz B. 2003. European Pharmacopoeia. Stability Testing: Photostability Testing of New Drug Substances and Products. Vol. 32nd edn. ICH. ICH. Richter A and Läer S. MD. Prolongation of survival in congestive cardiomyopathy by beta-receptor blockade. Hjalmarson A. Chromatogr. Lancet 1979. Stability studies of expired Tablets of metoprolol tartrate and propranolol hydrochloride.hydroxymetoprolol. Project Director. IFPMA: Geneva. Q1A (R2).B. B: Biomedical Sciences and Applications 1987. and propranolol in postmortem human fluid and tissue specimens via LC/APCI-MS. Waagstein F and Wallentin I. Hyderabad and Dr Ahmed Kamal. 736 wileyonlinelibrary. Part 1. M. Borkar et al. 33(13): 1904–1908. Yadav. metoprolol. 2005. International Conference on Harmonization. Journal of Separation Science 2010. 156: 106–117. Detection. India for supporting this work. The United States Pharmacopeial Convention: Rockville. Q1B. R. Balmér K. British Pharmacopoeia. IFPMA: Geneva. Proposed fragmentation mechanism for DP14 (m/z 236). Validation of analytical procedure: text and methodology. Ltd. 2009. 417(2): 357–365. Director. 66(6): 697–701. S. IFPMA: Geneva. Acknowledgments The authors thank Dr J. Swedberg K. Q2 (R1). Orszulak-Michalak D and Kurczewska U. Journal of Chromatography. wishes to thank the management of the United States Pharmacopeia Laboratory. Simultaneous RP-HPLC determination of sotolol. Elshoff JP. H NH m/z 98 m/z 91 NH m/z 72 O OH NH H H m/z 236 NH m/z 56 O -CH4 m/z 105 -C3H9N OH H O O H O -C2H2 O H m/z 177 m/z 151 -H 20 O m/z 159 -C2H2 O C H C H m/z 133 Scheme 11. Quantitation of atenolol. Content determination. Karwowski B. References Albers S. Forensic Science International 2006. Zhang YY. Biomedical Chromatography 2005. Jasińska M. 19(3): 202–207. Lagerström PO and Persson BA.