Research article

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

(wileyonlinelibrary.com) DOI 10.1002/bmc.1721

Identication 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, specic and reliable isocratic high-performance liquid chromatography combined with quadrupole time-ofight electrospray ionization tandem mass spectrometry (LC/Q-TOF-ESI-MS/MS) method has been developed and validated for the identication 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-specied 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 (4.6 250 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 specicity, linearity, accuracy and precision. Copyright 2011 John Wiley & Sons, Ltd. Keywords: metoprolol; LC-ESI-MS/MS; degradation products; accurate mass measurements

Introduction
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 uids and in the presence of other drugs (Balmr et al., 1987; Albers et al., 2005; Yilmaz et al., 2010; Baranowska and Wilczek, 2009). Although Jasiska et al. (2009) carried out stability studies on expired tablets, the study was limited to identication, 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 (AH 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.

Experimental
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: srini@iict.res.in
a

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 PharmacopeiaIndia Private Limited, Research and Development Laboratory, ICICI Knowledge Park, Turkapally, Shameerpet, Hyderabad, 500 078, India Abbreviations used: CID, collision induced dissociation; TOF, time-of-ight.

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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

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

OH

m/z 121

m/z 133

Scheme 1. Proposed fragmentation mechanism for metoprolol drug (m/z 268).

present study were purchased from Merck, (Mumbai, India). Ammonium formate, formic acid, sodium hydroxide, hydrochloric acid and hydrogen peroxide were obtained from Merck (Darmstadt, Germany). All reagents used were at least of analytical grade, except for methanol and acetonitrile. HPLC-grade water was obtained by passage through a Milli-QW system, Progard 2 (Millipore, Milford, MA, USA), and was used to prepare all solutions

Apparatus and equipment

Degradation studies were carried out in water bath equipped with a temperature controller. A controlled temperature oven (Mack Pharmatech Private Ltd, 830 V, Sr. no. 46/07-08) was used for solid-state thermal stress studies. A photostability chamber (Mack equipment, MK-10-PH, 230 V Phase) was used for the photodegradation study. The photostability

Table 1. 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 1.2 106 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

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R. M. Borkar et al.
chamber consisted of both UV and uorescent lamps. A calibrated lux meter and UV meter were used to measure energy. All pH measurement was done using a pH-meter (Metrohm Schweiz AG, 780 pH meter, Germany) with an Epson printer Lx-300 t. Other equipment used included a sonicator and a Sartorius balance (CD 225 D, 22308105 Germany). The analysis was performed on an Agilent 1200 series HPLC instrument (Agilent Technologies, USA). The HPLC system consisted of a quaternary pump, an on-line degasser, a diode-array detector, an autoinjector, a column oven and a computer system embedded with Chemstation software. The samples were separated on a Waters Symmetry C18 column (250 4.6 mm, particle size 5 mm). LC-MS analysis was carried out on an Agilent 1200 series HPLC instrument (Agilent Technologies, USA) coupled to a quadrupole time-of-ight mass spectrometer (Q-TOF LC/MS 6510 series classic G6510A, Agilent Technologies, USA) equipped with an electrospray ionization source. The data acquisition and processing were carried out using Mass Hunter workstation software. A splitter was placed before the electrospray ionization source, allowing entry of only 35% of the eluent. MSn experiments were performed using a quadrupole ion trap mass spectrometer (Thermo Finnigan, San Jose, CA, USA), equipped with an electrospray ionization source. The data acquisition and processing were under the control of Xcalibur software. conducted at 80  C with a drug concentration of 1 mg/mL. The oxidative degradation study was carried out with 15% H2O2 at room temperature for 25 days at a concentration of 1 mg/mL. Solid-state photolytic studies were carried out by exposing light to a thin layer (1 mm) of drug in a Petri dish to 1.2 106 lx h of uorescent light and 200 W h/m2 UV-A light in a photostability chamber (ICH, 1996). For thermal stress, the drug was kept at 100  C in the oven for 4 days. The optimized stressed conditions are outlined in Table 1. All stressed samples were withdrawn at suitable time intervals and diluted 10 times with mobile phase. All the solutions were ltered using 0.22 mm membrane lters before HPLC and LC-MS analysis.

Separation studies
The main objective of this work was to separate metoprolol and its degradations products. Initially, stressed sample solutions were subjected to analysis by a method involving a Waters symmetry C18 column (250 4.6 mm i.d.; 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 other conditions were, ow-rate 1.1 mL/min, detection wave length 225 nm and column temperature 25  C. However, good separation was not achieved, even with varying pH, ratio of mobile phase components and ow-rate, and changing the organic modier to acetonitrile. Several studies were carried out by changing ratio of acetonitrile until satisfactory resolution was obtained. The mobile phase was ltered through a 0.45 mm Chrom Tech Nylon-66 lter and degassed prior to use.

Stressed degradation studies

Stress degradation studies of metoprolol were carried out under hydrolysis (acid, base and neutral), oxidation, dry heat and photolytic conditions as per ICH (2003) guidelines. Acidic and basic hydrolysis was carried out in 2 M HCl, 1 M NaOH, for 24 and 48 h, respectively, whereas neutral hydrolysis was carried out in water for 48 h. 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 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; the capillary was set at 30003500 V; the skimmer was 60 V; and nitrogen was used as the drying (300  C; 9 L/min) and nebulizing (45 psi) gas. For collision induced dissociation (CID) experiments, keeping MS1 static, the precursor ion of interest was selected using the quadrupole analyzer and the product ions were analyzed using a time-of-ight (TOF) analyzer. Ultra high-purity nitrogen was used as the collision gas, and the pressure in the collision cell was maintained at 18 Torr. All the spectra were recorded under identical experimental conditions, and an average of 2025 scans. The ESI source conditions for MSn studies were: spray voltage, 5 kV; capillary voltage, 1520 V; capillary temperature, 200  C; tube lens offset

Table 2. Parameters of linear regression equation Parameter Calibration range (ng/mL) Correlation coefcient (r2) Slope Intercept SD of slope SD of intercept Value 1060 0.9998 13654 4590 94.3032 3672.59

Table 3. Data of intra-day and inter-day precision studies (n = 3) Concentration (ng/mL) 30 50 60 Intra-day precision, measured concentration (ng/mL), SD; RSD (%) 29.85 0.0394; 0.13 49.92 0.0345; 0.06 60.02 0.0354; 0.05 Inter-day precision, measured concentration (ng/mL), SD; RSD (%) 29.86 0.0542; 0.18 49.90 0.0374; 0.07 60.11 0.0527; 0.08

Table 4. Recovery data for metoprolol spiked into a mixture of stressed samples Spiked concentration (ng/mL) 25 30 35 Calculated spiked concentration (ng/mL), SD; RSD (%) 24.90 0.0417; 0.16 30.01 0.0444; 0.14 34.94 0.0397; 0.11 Recovery (%) 99.6 100.06 99.84

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Figure 1. (a) LC-ESI-MS total ion chromatograms (TIC) of acid degradation products; (b) LC-ESI-MS-TIC of neutral/photolysis/thermal degradation products; (c) LC-ESI-MS-TIC of base degradation products; (d) LC-ESI-MS-TIC of oxidation degradation products.

voltage, 20 V; sheath gas (N2) pressure, 30 psi; and helium used as damping gas. For the ion trap mass analyzer, the automatic gain control settings were 2 107 for a full-scan mass spectrum and 2 107 counts for a full-product ion mass spectrum with a maximum ion injection time

of 200 ms. In full-scan MS2 and MS3 modes, the precursor ion of interest was rst isolated by applying an appropriate waveform across the endcap electrodes of the ion trap to resonantly eject all trapped ions, except those ions of the m/z ratio of interest. The isolated ions were then

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Observed mass (Da) 268.1912 209.1161 153.0985 135.0801 250.1815 137.0967 167.1051 238.1811 226.1432 116.1092 58.0655 383.2911 60.0581 222.1471 236.1655 268.1907 209.1172 153.1910 135.0804 250.1802 137.0961 167.1067 238.1802 226.1438 116.1070 58.0651 383.2904 60.0570 222.1489 236.1645 2.18 4.66 6.12 1.11 2.62 2.16 4.16 2.58 0.91 4.12 2.12 3.15 3.22 5.12 3.11 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, 191, 159, 133, 218, 176, 226, 116, 57, 98, 74, 56, 121, 72 167, 149, 135, 121, 117, 105, 99, 73, 59, 57 153, 111, 69 250, 191, 149, 56, 218, 176, 98,165, 74, 56 179, 99, 151, 57, 133, 72, 158, 105, 123, 91 226, 159, 194, 165, 176, 133, 74, 121, 148, 116, 98 116, 74, 59, 56 383, 324, 306, 268, 232, 116 163, 149, 135, 99, 74, 58 177, 159, 151, 133, 105, 98, 91, 72, 56 R. M. Borkar et al.

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Table 5. Elemental compositions for metoprolol and its degradation products

Drug/degradation product

Rt (min)

Proposed formula

Copyright 2011 John Wiley & Sons, Ltd.

Drug DP1 DP2 DP3 DP4 DP5 DP6 DP7 DP8 DP9 DP10 DP11 DP12 DP13 DP14

10.3 2.9 3.3 3.8 4.6 5.0 5.8 6.2 6.8 8.5 12 15.2 17.0 18.0 18.8

C15H26NO3 C12H17O3 C9H13O2 C9H11O C15H24NO2 C9H13O C10H15O2 C14H24NO2 C12H20NO3 C6H14NO C3H8N C21H39N2O4 C3H8O C13H20NO2 C14H22NO2

Interaction product having m/z 383.

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Degradation products of metoprolol

Figure 2. (a) LC-ESI-MS/MS spectrum of [M + H] + ions (m/z 268) of metoprolol at 26 eV; (b) LC-ESI-MS/MS spectrum of [M + H]+ ions (m/z 209) of DP1 at 20 eV; (c) LC-ESI-MS/MS spectrum of [M + H] + ions (m/z 153) of DP2 at 16 eV; (d) LC-ESI-MS/MS spectrum of [M + H]+ ions (m/z 250) of DP4 at 25 eV; (e) LCESI-MS/MS spectrum of [M + H]+ ions (m/z 238) of DP7 at 32 eV, (f) LC-ESI-MS/MS spectrum of [M + H] + ions (m/z 226) of DP8 at 21 eV; (g) LC-ESI-MS/MS spectrum of [M + H]+ ions (m/z 116) of DP9 at 16 eV; (h) LC-ESI-MS/MS spectrum of [M + H]+ ions (m/z 383) of DP11 at 21 eV; (i) LC-ESI-MS/MS spectrum of [M + H]+ ions (m/z 222) of DP13 at 32 eV; (j) LC-ESI-MS/MS spectrum of [M + H]+ ions (m/z 236) of DP14 at 22 eV.

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R. M. Borkar et al.

Figure 2. (Continued).

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Degradation products of metoprolol

Figure 2. (Continued).

Table 6. Elemental compositions for daughter ion of metoprolol, 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) 268.1912 250.1815 191.1054 159.0814 133.0632 218.1532 176.1071 226.1432 116.1092 57.0336 98.0954 74.0611 56.0491 121.0641 72.0451 57.0336 Calculated mass (Da) 268.1907 250.1802 191.1067 159.0804 133.0648 218.1539 176.1070 226.1438 116.1070 57.0335 98.0964 74.0600 56.0495 121.0648 72.0444 57.0335 Error (ppm) 2.18 2.62 2.98 2.12 2.96 3.12 0.98 0.91 4.12 0.16 2.62 1.16 0.92 1.21 4.15 0.16 Proposed neutral loss H 2O C3H7NH2 CH3OH C2 H 2 CH3OH C3 H 6 CH3CH&dbond;CH2 C9H12O2 C12H21NO H 2O C3 H 6 H 2O C3H3NH2 C3 H 8 C12H21NO

Metoprolol

subjected to a supplementary a.c. signal to resonantly excite them and so cause CID. The collision energies used were 2035 eV. The excitation time used was 30 ms; all the spectra were recorded with an average of 2530 scans.

Results and discussion

Development and optimization of LC and LC-MS method Initially, metoprolol was analyzed on a C18 column (250 4.6 mm i.d.; particle size 5 mm) using 20 mM ammonium formate buffer (pH 3) and methanol in the ratio of 80:20 at a ow-rate of 1 mL/min. Under these conditions the peak shape of the drug was not satisfactory. Subsequent trials were made on a mixture of degraded samples by changing organic modier to

LC-MS/TOF studies on degradation products

Both the drug and degraded samples were investigated using LC-MS/ TOF mass spectrometry. The degradation products were analyzed by MS/MS, MSn fragmentation and accurate mass measurements.

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R. M. Borkar et al.

O O CH3 H3CO DP1 m/z 209

H OH

H H

H3CO DP2

m/z 153

H3CO DP3

m/z 135

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

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

H3C DP13

m/z 222 DP14

m/z 236

Scheme 2. Proposed structures of degradation products formed under various stress conditions.

O O CH 3 H m/z 73

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. Proposed fragmentation mechanism for DP1 (m/z 209).

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Degradation products of metoprolol acetonitrile. 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, pH 3): acetonitrilemethanol in the ratio of 85:15:5. The ow-rate was 1.1 mL/min and detection wave length was maintained at 225 nm. For LC-MS studies, same method was used as for HPLC, without replacement of buffer. The Q-TOF ESI source conditions were also optimized to obtain a good signal and high sensitivity. The conditions like drying gas ow, nebulizing gas ow, drying gas temperature, capillary voltage, 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. Method validation The stability-indicating method was validated for linearity, precision (inter-day, intra-day and intermediate precision), accuracy and specicity. The optimized LC-MS method was validated with respect to various parameters summarized in the ICH (2005) guidelines. To establish linearity and range, a stock solution containing 1 mg/mL metoprolol in mobile phase was diluted to yield solutions in the concentration range of 1060 ng/mL. The solutions were prepared and analyzed in triplicate. The response for the drug was linear in the investigated concentration (r2 = 0.9998) and the %RSD for each investigated concentration was <0.15%. The linearity data are given in Table 2. The

Table 7. Elemental compositions for daughter ions of DP1 (m/z 209), 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) 209.1161 167.1061 149.0582 135.0801 121.0652 117.0911 105.0681 99.0440 73.0266 59.0482 57.0336 153.0985 111.0812 69.0336 250.1815 191.1078 149.0812 56.0491 218.1536 176.1071 98.0954 166.1234 74.0712 Calculated mass (Da) 209.1172 167.1067 149.0597 135.0804 121.0648 117.0910 105.0699 99.0441 73.0284 59.0491 57.0335 153.1910 111.0804 69.0335 250.1802 191.1305 149.0835 56.0495 218.1539 176.1070 98.0964 166.1226 74.0964 Error (ppm) 4.66 2.11 4.92 1.11 5.23 1.11 4.10 0.68 5.11 4.26 0.16 6.12 4.22 0.12 2.62 2.91 3.12 0.92 0.11 0.98 2.62 3.14 4.62 Proposed neutral loss C2H2O CH3CH2OCH3 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. Proposed fragmentation mechanism for DP2 (m/z 153).

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R. M. Borkar et al. intra- and inter-day precisions were determined at three different concentrations, 30, 50 and 60 ng/mL, on the same day (n = 3) and consecutive days (n = 3). Table 3 shows that the %RSD for intraand inter-day precision was <0.15 and <0.20% respectively, indicating that the method was sufciently precise. Table 4 shows that recoveries of the added drug were obtained from the difference between peak areas of fortied and unfortied degraded samples. The specicity 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, and was also demonstrated by subjecting all the degradation samples to LC-MS. The mass detector showed an excellent purity for metoprolol and every degradation product, which unambiguously proves the specicity of the method. Oxidative stress. No signicant degradation was observed in 3% H2O2 after 48 h at room temperature; instead, extensive degradation was observed in 15% H2O2 after 25 days (Fig. 1d), resulting in eight degradation products (DP1, DP2, DP3, DP6, DP7, DP8, DP9 and DP11). Photolysis and solid-state studies. The drug was stable to light and thermal stress in the solid state, as shown in Fig.1(b). LC-MS/MS and MSn studies of metoprolol and its degradation products Fragmentation pathway of the protonated drug. The positiveion ESI mass spectrum of metoprolol shows an abundant [M + H]+ ion at m/z 268. The MS/MS spectrum (Fig. 2a; Johnson and Lewis, 2006) shows product ions at m/z 250 (loss of H2O), m/z 226 (loss of propene), m/z 218 (loss of methanol from m/z 250), m/z 191 (loss of propan-2-amine from m/z 250), m/z 176 (loss of propene from m/z 218), m/z 159 (loss of methanol from m/z 191), m/z 133 (loss of C2H2 from m/z 159), m/z 121 (protonated 4-vinylphenol), m/z 116 (loss of 4-(2-methoxyethyl)phenol), m/z 98 (loss of H2O from m/z 116), m/z 74 (protonated 3-aminoprop-1-en-2-ol), m/z 72 (protonated 1-iminopropan-2-one), m/z 57 (protonated methoxyethyne) and m/z 56 (protonated propa-1,2-dien-1-amine; Scheme 1). All these fragmentation pathways were conrmed by MSn experiments and accurate mass measurements (Table 6). MS/MS CID of degradation products. To characterize all the degradation products formed under various stress conditions, the CID of the protonated degradation products was carried out using Q-TOF and ion trap mass spectrometers. The proposed structures and elemental compositions of all the degradation products are shown in Scheme 2 and Table 5, respectively.

Degradation behavior The optimized LC-MS method is applicable for identifying the degradation products. The LC-ESI-MS total ion chromatograms obtained under various stress conditions are given in Fig. 1. A total of 14 degradation products were identied and characterized by tandem mass spectrometric analysis (LC-ESI-MS/MS). The degradation products are given different notations, viz. DP1-DP14 (Table 5). Hydrolysis. The drug showed an extensive degradation in 2 M HCl at 80  C (Fig. 1a) and after 4 days seven degradation products (DP3, DP4, DP6, DP10, DP12, DP13 and DP14) were formed. In neutral conditions, on heating the drug in water for 4 days at 80  C, no degradation product was formed (Fig. 1b), indicating that the drug is stable. In comparison to neutral stress conditions, two degradation products, DP5 and DP9, were observed on treatment of the drug in 1 M NaOH for 48 h at 80  C (Fig. 1c).

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

m/z 191 -C3H6 H O m/z 149 -C6H5O NH 2 m/z 56 NH 2

NH2

m/z 176

Scheme 5. Proposed fragmentation mechanism for DP4 (m/z 250).

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Degradation products of metoprolol DP1 ([M + H]+, m/z 209). The ESI-MS/MS spectrum of [M + H]+ ions (m/z 209) of DP1 (Rt = 2.9 min), shows product ions at m/z 167 (loss of C2H2O), m/z 149 (loss of CH3CH2OCH3), m/z 135 (loss of methanol from m/z 167), m/z 121 (loss of CO m/z 149), m/z 117 (loss of phenol), m/z 105 (protonated styrene), m/z 99 [protonated 1-(ethynyloxy)propan-2-one], m/z 73 (protonated 2-oxopropanal), m/z 57 (protonated methoxyethyne) and m/z 59 (C3H7O+; Fig. 2b, Scheme 3). These fragmentation pathways have been conrmed by MSn experiments and accurate mass measurements (Table 7). 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. All these data are consistent with the proposed structure 1-[4-(2methoxyethyl) phenoxy] propan-2-one proposed for DP1. DP2 ([M + H]+, m/z 153). The DP2 formed under oxidative stress conditions eluted at Rt 3.3 min. The molecular mass of DP2 was found to correspond to the molecular mass of impurity B (European and British pharmacopoeias). 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; Fig. 2c, Scheme 4). These characteristic fragment ions were highly compatible with the structure of 4-(2-methoxyethyl) phenol. The elemental compositions of all fragment ions were conrmed by accurate mass measurements (Table 7). DP4 ([M + H]+, m/z 250). The ESI-MS/MS spectrum of [M + H]+ ions (m/z 250) of DP4 (Rt = 4.6 min), was 18 Da less than the drug, suggesting a loss of water molecules from the protonated drug (m/z 268). The CID spectrum showed abundant product ions at m/ z 166 (loss of C5H8O) and m/z 98 (loss of C9H12O2; Fig. 2d, Scheme 5) and low-abundance ions at m/z 218 (loss of methanol), m/z 191 (loss of C3H7O), m/z 176 (loss of C3H6 from m/z 218), m/z 149 (loss of propene from m/z 191), m/z 74 (protonated N-methylpropan-2amine), m/z 56 (C3H6N+). All these fragmentation pathways were conrmed by MSn experiments. The formation of m/z 98 is indicative of the presence of the N-isopropylprop-1-en-1-amine group, which may be attached to phenoxy moiety. The formation of fragment ions at m/z 218, m/z 191, 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. 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

CH3

H CH2

O CH3

O
m/z 133

H CH

m/z 123

m/z 57

Scheme 6. Proposed fragmentation mechanism for DP7 (m/z 238).

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R. M. Borkar et al. and its fragment ions were conrmed by accurate mass measurements (Table 7). All these data are consistent with the proposed structure 3-[4-(2-methoxyethyl) phenoxy]-N-isopropylprop-1-en-1amine for DP4. DP7 ([M + H]+, m/z 238). The ESI-MS/MS spectrum of [M + H]+ ions (m/z 238) of DP7 (Rt = 6.2 min) was 30 Da less than the protonated drug, suggesting a neutral loss of formaldehyde from the protonated drug (m/z 268). The CID of protonated DP7 yielded abundant product ions at m/z 179 (loss of C3H7NH2), m/z 99 [protonated 1-(ethynyloxy) propan-2-one; Fig. 2e, Scheme 6], and low-abundance ions at m/z 159 (loss of C3H13NO), m/z 151 (loss of C2H4 from m/z 179 ), m/z 133 (loss of H2O from m/z 151), m/z 123 (loss of CO from m/z 151), m/z 57 (propan-2-one cation), m/z 105 (1-ethylbenzene cation), m/z 72 (N-methylpropan-2-amine cation) and m/z 91 (C7H+ 7 ). All these fragmentation pathways were conrmed by MSn experiments and accurate mass measurements (Table 8). As can be seen from Scheme 6, the fragment ions at m/z 179, m/z 159, m/z 133, m/z 123 and m/z 105 are highly compatiable with the structure 1-(4-ethylphenoxy)-3- (isopropyl amino) propan2-ol proposed for DP7. DP8 ([M + H] , m/z 226). The ESI-MS/MS spectrum of [M + H] ions (m/z 226) of DP8 (Rt = 6.8 min) displayed abundant product ions at m/z 121 (4-vinylphenol) and m/z 74 (1-aminopropan-2one; Fig. 2f, Scheme 7), and low-abundance ions at m/z 194 (loss of methanol), m/z 176 (loss of H2O from m/z 194), m/z 165 (loss of methanimine from m/z 194), m/z 159 (loss of methanol from m/z 191), m/z 148 (loss of C2H4 from m/z 176), m/z 133 (loss of C2H2 from m/z 159), m/z 116 (protonated 1-amino-3- (ethynyloxy) propan-2-ol), m/z 98 [protonated 3-(ethynyloxy) prop-1-en-1amine] and m/z 56 (protonated propa-1,2-dien-1-amine). These
+ +

fragmentation pathways were conrmed by MSn experiments. 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, respectively, and the fragment ions at m/z 98, 76, and 54 are diagnostic for the presence of 3-(ethynyloxy) prop-1-en-1amine moiety. The elemental compositions of DP8 and its fragment ions were conrmed by accurate mass measurements (Table 8). All these data are highly compatible and conrm the structure 1-[4-(2-methoxyethyl) phenoxy]-3-aminopropan-2-ol proposed for DP8. DP9 ([M + H]+, m/z 116). The DP9 formed under both oxidative and basic stress conditions eluted at an Rt of 8.5 min. The protonated DP9 yielded product ions at m/z 74 (loss of propene), m/z 56 (loss of H2O from m/z 74), m/z 57 (C3H5O+) and m/z 59 (protonated propan-2-one; Fig. 2g) The elemental compositions of DP9 and its fragment ions were been conrmed by accurate mass measurements (Table 9). It can be noted that DP9 corresponds to the complementary product ion of DP2 (Scheme 8). Based on these data, DP9 was identied as protonated 1-(isopropyl amino) propan-2-one. DP11 (m/z 383). Figure 2(h) shows the ESI-MS2 spectrum of [M + H]+ ions (m/z 383) of DP11, which eluted at an Rt of 15.2 min. The protonated DP11 yielded products ions at m/z 324 (loss of C3H7NH2), m/z 306 (loss of H2O from m/z 324), m/z 332 (loss of C6H5O from m/z 324), m/z 268 (protonated metoprolol) and m/z 116 [protonated 1-(isopropyl amino) propan-2-one]. These fragmentation pathways were conrmed by MSn experiments and accurate mass measurements. 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, as shown in Scheme 9. The MS/MS, MSn

Table 8. 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) 238.1811 179.1065 99.0445 151.0759 57.0336 133.0632 72.0811 91.0511 159.0814 123.0812 105.0698 226.1432 159.0814 194.1172 165.0929 176.1071 133.0632 74.0611 121.0672 148.0768 116.0711 98.0631 56.0491 Calculated mass (Da) 238.1802 179.1067 99.0441 151.0754 57.0335 133.0648 72.0808 91.0542 159.0804 123.0804 105.0699 226.1438 159.0804 194.1176 165.0910 176.1070 133.0648 74.0600 121.0648 148.0757 116.0706 98.0600 56.0495 Error (ppm) 2.58 0.16 0.24 1.89 0.16 2.96 3.14 4.22 2.12 3.98 0.16 0.91 2.12 1.10 4.16 0.98 2.96 1.16 5.16 4.98 3.92 5.32 0.92 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

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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

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. Proposed fragmentation mechanism for DP8 (m/z 226).

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. DP13 ([M + H]+, m/z 222). The DP13 formed under acidic stress conditions eluted at the Rt of 18.0 min. The ESI-MS2 spectrum of [M + H]+ ions (m/z 222) of DP13 displayed product ions at m/z 163 (loss of C3H7NH2), m/z 74 (protonated N-methylpropan2-amine), m/z 149 (loss of C4H9NH2), m/z 135 (loss of CO from m/z 163), m/z 99 (loss of C5H4 from m/z 163) and m/z 58 (propan-2-imine cation; Fig.2i, Scheme 10). The elemental compositions of protonated DP13 and its fragment ions were conrmed by accurate mass measurements (Table 9). All these data are in line with the structure 1-(p-tolyloxy)-3-(isopropyl amino) propan2-ol proposed for DP13. DP14 ([M + H]+, m/z 236). The ESI-MS/MS spectrum of [M + H]+ ions (m/z 236) of DP14 (Rt = 18.8 min) displayed abundant product ions at m/z 98 (protonated N-isopropylpropa-1,2-dien-1amine), m/z 177 (loss of propan-2-amine) and low-abundance ions at m/z 159 (loss of H2O from m/z 177), m/z 133 (loss of C2H2 from m/z 159), m/z 151 (loss of C2H2 from m/z 177),

m/z 105 (protonated styrene), m/z 91 (C7H7+), m/z 72 (C4H10N+) and m/z 56 (C3H6N+; Fig. 2j, Scheme 11). These fragmentation pathways were conrmed by MSn experiments. The molecular mass of DP14 was found to correspond to the molecular mass of impurity I (www.tlcpharmachem.com). The elemental compositions of DP14 and its fragments ions were conrmed by accurate mass measurements (Table 9). Based on the fragmentation pattern and accurate mass measurements, the possible structure 1-(4-vinylphenoxy)-3-(isopropyl amino) propan-2-ol can be assigned to DP14. DP3 ([M + H]+, m/z 135), DP5 ([M + H]+, m/z 137) and DP6 ([M + H]+, m/z 167). The DP5 formed under basic stress conditions, whereas DP6 formed under both acidic and oxidative stress conditions (Rt = 5.0 and 5.8 min, respectively). The ESI-MS of DP5 and DP6 showed abundant [M + H]+ ions at m/z 137 and m/z 167, respectively. The mass difference between DP5 and DP6 was CH2O (30 Da). Based on elemental compositions and accurate mass measurements (Table 5), DP5 and DP6 were identied as protonated 1-(2-methoxyethyl) benzene and protonated 1-methoxy-4-(2 methoxyethyl) benzene, respectively. Similarly, DP3 (Rt 3.8; m/z 135) was identied as protonated 1-(2-methoxyvinyl) benzene.

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R. M. Borkar et al. Table 9. Elemental compositions for daughter ions of DP9 (m/z 116), DP11 (m/z 383), 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) 116.1092 74.0611 59.0481 56.0491 57.0336 383.2911 324.2171 306.2053 232.1911 268.1912 116.1092 222.1471 163.0751 149.0591 135.0812 99.0412 74.0912 58.0655 236.1655 177.0911 159.0814 151.0759 133.0632 105.0681 98.0954 91.0511 72.0811 56.0491 Calculated mass (Da) 116.1070 74.0600 59.0491 56.0495 57.0335 383.2904 324.2169 306.2064 232.1907 268.1907 116.1070 222.1489 163.0754 149.0597 135.0804 99.0441 74.0964 58.0651 236.1645 177.0910 159.0804 151.0754 133.0648 105.0699 98.0964 91.0542 72.0808 56.0495 Error (ppm) 4.12 1.16 3.66 0.92 0.16 3.15 4.16 4.91 3.16 2.18 4.12 5.12 1.26 1.91 3.12 5.61 4.62 2.12 3.11 1.96 2.12 1.89 2.96 4.10 2.62 4.22 3.14 0.92 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

m/z 59 -H2O NH2 H

Scheme 8. Proposed fragmentation mechanism for DP9 (m/z 116).

DP10 ([M + H]+, m/z 58) and DP12 ([M + H]+, m/z 60). The DP10 and DP12 formed under acidic stress conditions with Rt of 12.0 and 17.0 min, respectively. Based on elemental

compositions and accurate mass measurements (Table 5), DP10 and DP12 were identied as protonated propan-2-imine and protonated propan-2-amine, respectively.

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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. Proposed fragmentation mechanism for DP11 (m/z 383).

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

CH 2

Scheme 10. Proposed fragmentation mechanism for DP13 (m/z 222).

Conclusions
Stress degradation studies on metoprolol, carried out according to ICH guidelines, provided information on the degradation behavior of the metoprolol under the conditions of hydrolysis and oxidation. 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. The drug showed extensive degradation in acid, base hydrolysis and oxidative stress, while it was stable to neutral, thermal and photolytic stress conditions. A total of 14 degradation products were characterized with the help of the MS/MS, MSn experiments combined with accurate mass measurements of fragment ions and precursors. Of these degradation products, DP2 (m/z 153) and DP14 (m/z 236) matched impurities B and I, respectively.

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R. M. Borkar et al.

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

O O

H O -C2H2

m/z 177 m/z 151 -H 20 O m/z 159 -C2H2 O C H C H

m/z 133

Scheme 11. Proposed fragmentation mechanism for DP14 (m/z 236).

Acknowledgments
The authors thank Dr J. S. Yadav, Director, IICT, Hyderabad and Dr Ahmed Kamal, Project Director, NIPER, Hyderabad for facilities and their cooperation. B.R. is thankful to DST for the award of a Junior Research Fellowship. R.B. wishes to thank the management of the United States Pharmacopeia Laboratory, India for supporting this work.

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British Pharmacopoeia, medicinal and pharmaceutical substances. 2009; I and II 39333936. European Pharmacopoeia, 5thEdition, 2005; 5: 20322033. ICH. Stability Testing: Photostability Testing of New Drug Substances and Products. Q1B. International Conference on Harmonization, IFPMA: Geneva, 1996. ICH. Stability Testing of New Drug Substances and Products. Q1A (R2). International Conference on Harmonization, IFPMA: Geneva, 2003. ICH. Q2 (R1). Validation of analytical procedure: text and methodology. International Conference on Harmonization, IFPMA: Geneva, 2005. Jasiska M, Karwowski B, Orszulak-Michalak D and Kurczewska U. Stability studies of expired Tablets of metoprolol tartrate and propranolol hydrochloride. Part 1. Content determination. Acta Poloniae Pharmaceutica 2009; 66(6): 697701. Johnson RD and Lewis RJ. Quantitation of atenolol, metoprolol, and propranolol in postmortem human uid and tissue specimens via LC/APCI-MS. Forensic Science International 2006; 156: 106117. Swedberg K, Hjalmarson A, Waagstein F and Wallentin I. Prolongation of survival in congestive cardiomyopathy by beta-receptor blockade. Lancet 1979; 1: 13741376. United States Pharmacopeia. 32nd edn, Vol. 3. The United States Pharmacopeial Convention: Rockville, MD, 2009; 29632970. Yilmaz B, Asci A and Arslan S. Detection. Journal of Separation Science 2010; 33(13): 19041908.

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