acterization of stressedf metoprolol usingd MSn experiments

ivasa,b*, Prashant Patelb

an-Tpation, photolysis and thermal stress, as per ICH-specied condi-e and hydrolysis (acid and base) stress conditions. However, itns.roragfraF

(2-methoxyethyl) phenoxy]-2-propanol. In the literature, many

Research article

Published online in Wiley Online Library: 12 October 2011

720LC and LC-MS methods have been reported for the analysis ofdrug in biological uids and in the presence of other drugs (Balmret al., 1987; Albers et al., 2005; Yilmaz et al., 2010; Baranowskaand Wilczek, 2009). Although Jasiska et al. (2009) carried outstability studies on expired tablets, the study was limited toidentication, characterization and the degradation pathway ofthe drug. The drug substance monograph on metoprolol inthe European Pharmacopoeia (2005) and British Pharmacopoeia(2009) lists nine impurities (AH and J). Of these, four are alsomentioned as related substances in the drug monograph inthe United States Pharmacopeia (2009). Impurity I is mentionedon the TLC pharmachem website (http://www.tlcpharmachem.com/tlc_item.php?upc=M-0812&li=&sub=).

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 ChemicalTechnology, Hyderabad, 500 607, India

b National Institute of Pharmaceutical Education and Research, Balanagar,Hyderabad, 500 037, India

cachieved 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, respec-tively. The LC-MSmethodwas validatedwith respect to specicity, linearity, accuracy and precision. Copyright 2011 JohnWiley& Sons, Ltd.

Keywords: metoprolol; LC-ESI-MS/MS; degradation products; accurate mass measurements

IntroductionMetoprolol belongs to the class of selective b1 blocker receptorsused in the treatment of several cardiovascular diseases,especially hypertension. It has little or no effect on b2 blockerreceptors except in high doses. Treatment of heart failure byb-adrenergic blocking agent has been intensely investigated(Swedberg et al., 1979).

Chemically, metoprolol (Scheme 1) is 1-(iso-propylamino)-3-[4

thermal and photolysis. The resultant solutions were subjected tooptimized LC-MS, MS/MS, MSn and accurate mass measurementsto establish the fragmentation pattern of the drug and its degrada-tion products.

Experimental

Drug and reagentsIdentication and chardegradation products oLC/Q-TOF-ESI-MS/MS anRoshan M. Borkara,b, B. Rajua, R. Srinand Satheesh Kumar Shettyc

ABSTRACT: A rapid, specic and reliable isocratic high-performight electrospray ionization tandem mass spectrometry (LC/Qthe identication and characterization of stressed degradationwas subjected to hydrolysis (acidic, alkaline and neutral), oxidtions. The drug showed extensive degradation under oxidativwas stable to thermal, neutral and photolysis stress conditiochromatographic separation of the drug and its degradation pcharacterize degradation products, initially the mass spectral fof MS/MS, MSn and accurate mass measurements. Similarly,products were established by subjecting them to LC-MS/QTO

Received 29 June 2011, Accepted 5 September 2011

(wileyonlinelibrary.com) DOI 10.1002/bmc.1721The main aim of the present study was to investigate thecomplete degradation behavior of the drug and to characterizethe degradation products. This was done by exposing the drugto ICH-recommended stress conditions of hydrolysis, oxidation,

Biomed. Chromatogr. 2012; 26: 720736 Copyright 2011 JohnA total of 14 degradation products were observed and theducts was achieved on a C18 column (4.6 250mm, 5mm). Tomentation pathway of the drug was established with the helpgmentation pattern and accurate masses of the degradationanalysis. Structure elucidation of degradation products wasce liquid chromatography combined with quadrupole time-of-OF-ESI-MS/MS) method has been developed and validated forroducts of metoprolol. Metoprolol, an anti-hypertensive drug,United States PharmacopeiaIndia Private Limited, Research and DevelopmentLaboratory, ICICI Knowledge Park, Turkapally, Shameerpet, Hyderabad, 500 078,India

Abbreviations used: CID, collision induced dissociation; TOF, time-of-ight.

Wiley & Sons, Ltd.

OHN

OH

H3CO m/z 268

OHN

3CO

-H2O

O NH2

OH

H3CO

-CH3-CH=CH2

m/z 226

HN

OH

m/z 116

HN

O

m/z 116

H

H

H

HH

NH

OH

m/z 72

CH

C

OCH3 H

m/z 57

OHO

-H20 -C3H6

-C3H8

-C12H21NO2

Degradation products of metoprololHNH2

OH

m/z 74

HN

m/z 98H

-H20

Hpresent study were purchased from Merck, (Mumbai, India). Ammoniumformate, formic acid, sodium hydroxide, hydrochloric acid and hydrogenperoxide were obtained from Merck (Darmstadt, Germany). All reagentsused were at least of analytical grade, except for methanol andacetonitrile. HPLC-grade water was obtained by passage through aMilli-QW system, Progard 2 (Millipore, Milford, MA, USA), and was usedto prepare all solutions

NH2

m/z 56-C3

H

m/z 121

Scheme 1. Proposed fragmentation mechanism for metoprolol drug (m/z 2

Table 1. Optimized Stress conditions

Stress condition

HydrolysisAcid 2 M HClBase 1 M NaOHNeutral H2OOxidation 15 % H2O2PhotolysisFluorescent light 1.2 106 lx hUltra-violet light 200Wh/m2

Thermal 100 C

Biomed. Chromatogr. 2012; 26: 720736 Copyright 2011 Johnm/z 250

O

-C3H7NH2

OHN

HH

-CH3OHApparatus and equipment

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

H3COm/z 191

O

m/z 159

-CH3OH

O

m/z 133

-C2H2

m/z 218

O NH2

m/z 176

H6

H

H

OH H

-C3H3NH2

H

68).

Exposure Duration

80 C 24 h80 C 48 h80 C 49 hRoom temperature 24 days

Photostability chamberPhotostability chamberOven 4 days

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721

chamber consisted of both UV and uorescent lamps. A calibrated luxmeter and UV meter were used to measure energy. All pH measurementwas done using a pH-meter (Metrohm Schweiz AG, 780 pH meter,Germany) with an Epson printer Lx-300 t. Other equipment used includeda 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 quaternarypump, an on-line degasser, a diode-array detector, an autoinjector, acolumn oven and a computer system embedded with Chemstationsoftware. The samples were separated on a Waters Symmetry C18 column(250 4.6mm, particle size 5mm).

LC-MS analysis was carried out on an Agilent 1200 series HPLC instru-ment (Agilent Technologies, USA) coupled to a quadrupole time-of-ightmass spectrometer (Q-TOF LC/MS 6510 series classic G6510A, AgilentTechnologies, USA) equipped with an electrospray ionization source.The data acquisition and processing were carried out using Mass Hunterworkstation software. A splitter was placed before the electrospray ioni-zation source, allowing entry of only 35% of the eluent.

MSn experiments were performed using a quadrupole ion trap massspectrometer (Thermo Finnigan, San Jose, CA, USA), equipped with anelectrospray ionization source. The data acquisition and processing wereunder the control of Xcalibur software.

Stressed degradation studies

Stress degradation studies of metoprolol were carried out under hydroly-sis (acid, base and neutral), oxidation, dry heat and photolytic conditionsas per ICH (2003) guidelines. Acidic and basic hydrolysis was carried out in2 M HCl, 1 M NaOH, for 24 and 48 h, respectively, whereas neutral hydroly-sis was carried out in water for 48 h. All the hydrolytic studies were

conducted at 80 C with a drug concentration of 1mg/mL. The oxidativedegradation study was carried out with 15% H2O2 at room temperaturefor 25 days at a concentration of 1mg/mL. Solid-state photolytic studieswere carried out by exposing light to a thin layer (1mm) of drug in a Petridish to 1.2 106 lx h of uorescent light and 200Wh/m2 UV-A light in aphotostability chamber (ICH, 1996). For thermal stress, the drug was keptat 100 C in the oven for 4 days. The optimized stressed conditions areoutlined in Table 1. All stressed samples were withdrawn at suitable timeintervals and diluted 10 times with mobile phase. All the solutions wereltered using 0.22mm membrane lters before HPLC and LC-MS analysis.

Separation studies

The main objective of this work was to separate metoprolol and its degra-dations products. Initially, stressed sample solutions were subjected toanalysis by a method involving a Waters symmetry C18 column(250 4.6mm i.d.; particle size 5 mm) and a mobile phase comprising amixture of 20mM ammonium formate (pH adjusted to 3 by formic acid)and methanol. The other conditions were, ow-rate 1.1mL/min, detec-tion wave length 225 nm and column temperature 25 C. However, goodseparation was not achieved, even with varying pH, ratio of mobilephase components and ow-rate, and changing the organic modier to

and the pressure in the collision cell was maintained at 18 Torr. All the

3)

sur; RS

365

f s

co

24.30.34.

Table 2. Parameters of linear regression equation

SD of intercept 3672.59

R. M. Borkar et al.

722Table 3. Data of intra-day and inter-day precision studies (n=

Concentration(ng/mL)

Intra-day precision, meaconcentration (ng/mL), SD

30 29.85 0.0394; 0.150 49.92 0.0345; 0.060 60.02 0.0354; 0.0

Table 4. Recovery data for metoprolol spiked into a mixture o

Spiked concentration (ng/mL) Calculated spiked

253035Parameter Value

Calibration range (ng/mL) 1060Correlation coefcient (r2) 0.9998Slope 13654Intercept 4590SD of slope 94.3032Copyright 2011 Johnwileyonlinelibrary.com/journal/bmctressed samples

ncentration (ng/mL), SD; RSD (%) Recovery (%)

90 0.0417; 0.16 99.601 0.0444; 0.14 100.0694 0.0397; 0.11 99.84spectra were recorded under identical experimental conditions, and anaverage 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

edD (%)

Inter-day precision, measuredconcentration (ng/mL), SD; RSD (%)

29.86 0.0542; 0.1849.90 0.0374; 0.0760.11 0.0527; 0.08acetonitrile. Several studies were carried out by changing ratio ofacetonitrile until satisfactory resolution was obtained. The mobile phasewas ltered through a 0.45mm Chrom Tech Nylon-66 lter and degassedprior to use.

MS/MS and MSn studies of the drug

The fragmentation pathway of metoprolol was established by carryingout TOF-MS/MS and MSn studies in positive-ion ESI mode. The typicalQ-TOF operating source conditions for MS scan of metoprolol in thismode were optimized as follows: the fragmentor voltage was set at80 V; the capillary was set at 30003500 V; the skimmer was 60 V; andnitrogen was used as the drying (300 C; 9 L/min) and nebulizing(45 psi) gas. For collision induced dissociation (CID) experiments, keepingMS1 static, the precursor ion of interest was selected using the quadru-pole analyzer and the product ions were analyzed using a time-of-ight(TOF) analyzer. Ultra high-purity nitrogen was used as the collision gas,Biomed. Chromatogr. 2012; 26: 720736Wiley & Sons, Ltd.

Degradation products of metoprololvoltage, 20 V; sheath gas (N2) pressure, 30 psi; and helium used as damp-ing gas. For the ion trap mass analyzer, the automatic gain controlsettings were 2 107 for a full-scan mass spectrum and 2 107 countsfor a full-product ion mass spectrum with a maximum ion injection time

Figure 1. (a) LC-ESI-MS total ion chromatograms (TIC) of acid degradationproducts; (c) LC-ESI-MS-TIC of base degradation products; (d) LC-ESI-MS-TIC

Biomed. Chromatogr. 2012; 26: 720736 Copyright 2011 Johnof 200ms. In full-scan MS2 and MS3 modes, the precursor ion of interestwas rst isolated by applying an appropriate waveform across the end-cap electrodes of the ion trap to resonantly eject all trapped ions, exceptthose ions of the m/z ratio of interest. The isolated ions were then

products; (b) LC-ESI-MS-TIC of neutral/photolysis/thermal degradationof oxidation degradation products.

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Table

5.Elem

entalc

ompo

sitio

nsformetop

rolola

ndits

degrad

ationprod

ucts

Drug/de

grad

ation

prod

uct

R t(m

in)

Prop

osed

form

ula

Observe

dmass(Da)

Calcu

lated

mass(Da)

Error(ppm

)Prop

osed

neutralloss

MS/MSfrag

men

tions

Drug

10.3

C15H26NO3

268.19

1226

8.19

072

.18

25

0,19

1,15

9,13

3,21

8,17

6,22

6,11

6,57

,98,

74,5

6,12

1,72

DP1

2.9

C12H17O3

209.11

6120

9.11

724

.66

C3H7NH2

167,

149,

135,

121,

117,

105,

99,7

3,59

,57

DP2

3.3

C9H13O2

153.09

8515

3.19

106

.12

C6H13NO

153,

111,

69DP3

3.8

C9H11O

135.08

0113

5.08

041.11

C6H15NO2

DP4

4.6

C15H24NO2

250.18

1525

0.18

022

.62

H2O

250,

191,

149,

56,2

18,1

76,9

8,16

5,74

,56

DP5

5.0

C9H13O

137.09

6713

7.09

612

.16

C6H13NO2

DP6

5.8

C10H15O2

167.10

5116

7.10

674.16

C5H11NO

DP7

6.2

C14H24NO2

238.18

1123

8.18

022

.58

CH2O

179,

99,1

51,5

7,13

3,72

,158

,105

,123

,91

DP8

6.8

C12H20NO3

226.14

3222

6.14

380.91

C3H6

226,

159,

194,

165,

176,

133,

74,1

21,1

48,1

16,9

8DP9

8.5

C6H14NO

116.10

9211

6.10

704

.12

C9H12O2

116,

74,5

9,56

DP10

12C3H8N

58.065

558

.065

12

.12

C12H18O3

DP11

15.2

C21H39N2O4

383.29

1138

3.29

043

.15

IN*

383,

324,

306,

268,

232,

116

DP12

17.0

C3H8O

60.058

160

.057

03

.22

C12H18NO2

DP13

18.0

C13H20NO2

222.14

7122

2.14

895.12

C2H6O

163,

149,

135,

99,7

4,58

DP14

18.8

C14H22NO2

236.16

5523

6.16

453

.11

CH4O

177,

159,

151,

133,

105,

98,9

1,72

,56

a Interactio

nprod

uctha

ving

m/z

383.

R. M. Borkar et al.

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724

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 at20 eV; (c) LC-ESI-MS/MS spectrum of [M+H] + ions (m/z 153) ofDP2 at 16 eV; (d) LC-ESI-MS/MS spectrum of [M+H]

+ ions (m/z 250) of DP4 at 25 eV; (e) LC-ESI-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/MSspectrum 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.

Degradation products of metoprolol

Biomed. Chromatogr. 2012; 26: 720736 Copyright 2011 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/bmc

725

Figure 2. (Continued).

R. M. Borkar et al.

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726

Degradation products of metoprololsubjected to a supplementary a.c. signal to resonantly excite them andso cause CID. The collision energies used were 2035 eV. The excitationtime used was 30ms; all the spectra were recorded with an average of2530 scans.

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 byMS/MS, MSn fragmentation and accurate mass measurements.

Figure 2. (Co

Table 6. Elemental compositions for daughter ion of metoprolol,

Drug Proposed formula Observed mass (Da) Ca

C15H26NO3 268.1912C15H24NO2 250.1815

Metoprolol C12H15O2 191.1054C11H11O 159.0814C9H9O 133.0632C14H20NO 218.1532C11H14NO 176.1071C12H20NO3 226.1432C6H14NO 116.1092C3H5O 57.0336C6H12N 98.0954C3H8NO 74.0611C3H6N 56.0491C8H9O 121.0641C3H6NO 72.0451C3H5O 57.0336

Biomed. Chromatogr. 2012; 26: 720736 Copyright 2011 JohnResults and discussion

Development and optimization of LC and LC-MS method

Initially, metoprolol was analyzed on a C18 column (250 4.6mmi.d.; particle size 5 mm) using 20mM ammonium formate buffer(pH 3) and methanol in the ratio of 80:20 at a ow-rate of1mL/min. Under these conditions the peak shape of the drugwas not satisfactory. Subsequent trials were made on a mixtureof degraded samples by changing organic modier to

ntinued).

m/z 268

lculated mass (Da) Error (ppm) Proposed neutral loss

268.1907 2.18 250.1802 2.62 H2O191.1067 2.98 C3H7NH2159.0804 2.12 CH3OH133.0648 2.96 C2H2218.1539 3.12 CH3OH176.1070 0.98 C3H6226.1438 0.91 CH3CH&dbond;CH2116.1070 4.12 C9H12O257.0335 0.16 C12H21NO98.0964 2.62 H2O74.0600 1.16 C3H656.0495 0.92 H2O

121.0648 1.21 C3H3NH272.0444 4.15 C3H857.0335 0.16 C12H21NO

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727

OCH3

O

H3CO m/z 209

HOH

H3COm/z 153

CH3

HN

O

m/z 116

H

OHN

H3CO m/z 250

H

H3COm/z 135

m/z 137H3CO

OCH3

H

H

m/z 167

OHN

OH H

m/z 238

O NH2

OH

H3CO m/z 226

HN

H

m/z 58

OHN

OH

H3CO

CH2

HOHN

m/z 383 m/z 60

OHN

OH

H3C m/z 222

O

OH HNH

m/z 236

H2NH

DP1 DP2 DP3

DP4 DP5DP6

DP7 DP8 DP9

DP10DP11

DP12

DP13 DP14

H

H3CO

H

H

H

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

OCH3

O

H3CO

OCH3

H3CO

m/z 209

m/z 167

OCH3

O

m/z 149

OCH3

H2Cm/z 135

O

m/z 121

H2C

m/z 105

OCH3

OCH3HC m/z 57

CH2

m/z 59

HC OCH3

O

m/z 99

OCH3

O

m/z 73

m/z 117

H

H

H

H

H

H

H

CH3

- CO

H

-CH3-CH2 OCH3

-CH3OH

-CH2O

-C2H2O

-C7H10O

-C9H12O

-C6H4O

-C7H8O

-C7H10O

Scheme 3. Proposed fragmentation mechanism for DP1 (m/z 209).

R. M. Borkar et al.

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728

acetonitrile. The best separation with good peak shape wasachieved on the same column at 25 C using a mobile phasecomposed of an ammonium formate buffer (20mM, pH 3):acetonitrilemethanol in the ratio of 85:15:5. The ow-rate was1.1mL/min and detection wave length was maintained at 225nm.

For LC-MS studies, same method was used as for HPLC,without replacement of buffer. The Q-TOF ESI source conditionswere also optimized to obtain a good signal and high sensitivity.The conditions like drying gas ow, nebulizing gas ow, dryinggas temperature, capillary voltage, spray voltage and skimmervoltage were optimized to maximize the ionization in the sourceand sensitivity even at a very low concentration to identify andcharacterize the degradation products.

Method validation

The stability-indicating method was validated for linearity, preci-sion (inter-day, intra-day and intermediate precision), accuracyand specicity. The optimized LC-MS method was validated withrespect to various parameters summarized in the ICH (2005)guidelines. To establish linearity and range, a stock solutioncontaining 1mg/mL metoprolol in mobile phase was dilutedto yield solutions in the concentration range of 1060 ng/mL.The solutions were prepared and analyzed in triplicate. Theresponse for the drug was linear in the investigated concentra-tion (r2 = 0.9998) and the %RSD for each investigated concentra-tion was

intra- and inter-day precisions were determined at three differentconcentrations, 30, 50 and 60 ng/mL, on the same day (n=3) andconsecutive days (n=3). Table 3 shows that the %RSD for intra-and inter-day precision was

DP1 ([M+H]+, m/z 209). The ESI-MS/MS spectrum of [M+H]+

ions (m/z 209) of DP1 (Rt = 2.9min), shows product ions atm/z 167 (loss of C2H2O), m/z 149 (loss of CH3CH2OCH3), m/z135 (loss of methanol from m/z 167), m/z 121 (loss of CO m/z149), m/z 117 (loss of phenol), m/z 105 (protonated styrene),m/z 99 [protonated 1-(ethynyloxy)propan-2-one], m/z 73 (pro-tonated 2-oxopropanal), m/z 57 (protonated methoxyethyne)and m/z 59 (C3H7O

+; Fig. 2b, Scheme 3). These fragmentationpathways have been conrmed by MSn experiments and accu-rate mass measurements (Table 7). The formation of fragmentions at m/z 57 and m/z 59 is indicative of the presence ofmethoxyethyl group while fragment ions at m/z 121 and m/z149 authenticate the presence of phenoxy moiety in DP1. Allthese data are consistent with the proposed structure 1-[4-(2-methoxyethyl) phenoxy] propan-2-one proposed for DP1.

DP2 ([M+H]+, m/z 153). The DP2 formed under oxidative

stress conditions eluted at Rt 3.3min. The molecular mass ofDP2 was found to correspond to the molecular mass of impurityB (European and British pharmacopoeias). The [M+H]+ ion ofDP2 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 highlycompatible with the structure of 4-(2-methoxyethyl) phenol.The elemental compositions of all fragment ions were conrmedby 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 = 4.6min), was 18Da 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) andm/z 98 (loss of C9H12O2; Fig. 2d, Scheme 5)and low-abundance ions atm/z 218 (loss of methanol),m/z 191 (lossof C3H7O), m/z 176 (loss of C3H6 from m/z 218), m/z 149 (loss ofpropene from m/z 191), m/z 74 (protonated N-methylpropan-2-amine), m/z 56 (C3H6N

+). All these fragmentation pathways wereconrmed byMSn experiments. The formation ofm/z 98 is indicativeof the presence of the N-isopropylprop-1-en-1-amine group, whichmay be attached to phenoxy moiety. The formation of fragmentions at m/z 218, m/z 191, m/z 176 and m/z 149 points to thepresence of phenoxy-N-isopropylprop-1-en-1-amine moiety in thestructure of DP4. The elemental compositions of protonated DP4

O NHOH

H3C m/z 238

OCH2

OH

H C m/z 179

C

CH2NH

m/z 72

H3Cm/z 105

O C

m/z 159

H2C

H

H

m/z 91

-C6H15NO2

H

-C3H13NO

-C3H7NH2

O

Degradation products of metoprolol

73

H3CHC O

O

CH3m/z 99

O CH3

m/z 123

H

H

-C

-C6H8

Scheme 6. Proposed fragmentation mechanism for DP (m/z 238).7

Biomed. Chromatogr. 2012; 26: 720736 Copyright 2011 JohnOO

m/z 179CH3

OO

CH3m/z 151

H2

O

CH3m/z 57

OCH

m/z 133

-H20

H

H

H

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31

and its fragment ions were conrmed by accurate mass measure-ments (Table 7). All these data are consistent with the proposedstructure 3-[4-(2-methoxyethyl) phenoxy]-N-isopropylprop-1-en-1-amine for DP4.

DP7 ([M+H]+, m/z 238). The ESI-MS/MS spectrum of [M+H]+

ions (m/z 238) of DP7 (Rt = 6.2min) was 30Da less than the pro-tonated drug, suggesting a neutral loss of formaldehyde fromthe protonated drug (m/z 268). The CID of protonated DP7yielded 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 ofC3H13NO), 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 (C7H7

+). Allthese fragmentation pathways were conrmed by MSn experi-ments and accurate mass measurements (Table 8). As can beseen 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 withthe structure 1-(4-ethylphenoxy)-3- (isopropyl amino) propan-2-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 = 6.8min) displayed abundant productions at m/z 121 (4-vinylphenol) and m/z 74 (1-aminopropan-2-one; Fig. 2f, Scheme 7), and low-abundance ions at m/z 194 (lossof methanol), m/z 176 (loss of H2O from m/z 194), m/z 165 (lossof methanimine from m/z 194), m/z 159 (loss of methanol from

fragmentation pathways were conrmed by MSn experiments.The characteristic fragment ions at m/z 116 and 121 are indicativeof the presence of 1-aminopropan-2-ol and phenoxy groups inDP8, respectively, and the fragment ions at m/z 98, 76, and 54are diagnostic for the presence of 3-(ethynyloxy) prop-1-en-1-amine moiety. The elemental compositions of DP8 and its frag-ment ions were conrmed by accurate mass measurements(Table 8). All these data are highly compatible and conrm thestructure 1-[4-(2-methoxyethyl) phenoxy]-3-aminopropan-2-olproposed for DP8.

DP9 ([M+H]+,m/z 116). TheDP9 formed under both oxidative

and basic stress conditions eluted at an Rt of 8.5min. The proton-ated DP9 yielded product ions atm/z 74 (loss of propene),m/z 56(loss of H2O from m/z 74), m/z 57 (C3H5O

+) and m/z 59 (proton-ated propan-2-one; Fig. 2g) The elemental compositions of DP9and its fragment ions were been conrmed by accurate massmeasurements (Table 9). It can be noted that DP9 correspondsto the complementary product ion of DP2 (Scheme 8). Basedon these data, DP9 was identied as protonated 1-(isopropylamino) 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 of15.2min. The protonated DP11 yielded products ions at m/z324 (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 (protonatedmetoprolol) and m/z 116 [protonated 1-(isopropyl amino)propan-2-one]. These fragmentation pathways were conrmed

z 2

a)

R. M. Borkar et al.

732m/z 191), m/z 148 (loss of C2H4 from m/z 176), m/z 133 (loss ofC2H2 from m/z 159), m/z 116 (protonated 1-amino-3- (ethynyloxy)propan-2-ol), m/z 98 [protonated 3-(ethynyloxy) prop-1-en-1-amine] and m/z 56 (protonated propa-1,2-dien-1-amine). These

Table 8. Elemental compositions for daughter ions of DP7 (m/

Degradation product Proposed Formula Observed mass (D

DP7 C14H24NO2 238.1811C11H15O2 179.1065C5H7O2 99.0445C9H11O2 151.0759C3H5O 57.0336C9H9O 133.0632C4H10N 72.0811C7H7 91.0511C11H11O 159.0814C8H11O 123.0812C8H9 105.0698

DP8 C12H20NO3 226.1432C11H12O 159.0814C11H16NO2 194.1172C10H13O2 165.0929C11H14NO 176.1071C9H9O 133.0632C3H8NO 74.0611C8H9O 121.0672C9H10NO 148.0768C5H10NO2 116.0711C5H10NO2 98.0631C9H11O2 56.0491Copyright 2011 Johnwileyonlinelibrary.com/journal/bmcby MSn experiments and accurate mass measurements. Theformation of fragment ions at m/z 268 and m/z 116 indicatesthat DP11 can be formed by the combination of corre-sponding moieties, as shown in Scheme 9. The MS/MS, MSn

38) and DP8 (m/z 226)

Calculated mass (Da) Error (ppm) Proposed neutral loss

238.1802 2.58 179.1067 0.16 C3H7NH299.0441 0.24 C6H8

151.0754 1.89 C2H457.0335 0.16 C6H6O

133.0648 2.96 H2O72.0808 3.14 C10H14O291.0542 4.22 C7H17NO2

159.0804 2.12 C3H13NO123.0804 3.98 CO105.0699 0.16 C6H15NO2226.1438 0.91 159.0804 2.12 NH3194.1176 1.10 CH4O165.0910 4.16 CHNH2176.1070 0.98 H2O133.0648 2.96 C2H274.0600 1.16 C8H8O

121.0648 5.16 C3H5ONH2148.0757 4.98 C2H4116.0706 3.92 C7H10O98.0600 5.32 H2O56.0495 0.92 H2OBiomed. Chromatogr. 2012; 26: 720736Wiley & Sons, Ltd.

Om/

19

z 17

H10O

3OH

Degradation products of metoprololH3CO

O

m/z

O

m/O

OH

OH

m/z 121

NH2

O

m/z 74

H

H

H

O

OH

m/z 116

NH2m/z 56

H

-H2OCH2=NH

-C3H5ONH2

-C8H8O

-C7-H2O

-CHfragment ions and elemental compositions derived fromaccurate mass measurements (Table 9) are consistent withthe proposed structure 1-({2-hydroxy-3-[4-(2-methoxyethyl)phenoxy] propyl} (isopropyl) amino)-3-(isopropyl amino) propan-2-ol for DP11.

DP13 ([M+H]+,m/z 222). The DP13 formed under acidic stress

conditions eluted at the Rt of 18.0min. The ESI-MS2 spectrum of

[M+H]+ ions (m/z 222) of DP13 displayed product ions atm/z 163 (loss of C3H7NH2), m/z 74 (protonated N-methylpropan-2-amine), m/z 149 (loss of C4H9NH2), m/z 135 (loss of CO fromm/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 compo-sitions of protonated DP13 and its fragment ions were conrmedby accurate mass measurements (Table 9). All these data are inline with the structure 1-(p-tolyloxy)-3-(isopropyl amino) propan-2-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 = 18.8min) displayed abundant prod-uct ions at m/z 98 (protonated N-isopropylpropa-1,2-dien-1-amine), m/z 177 (loss of propan-2-amine) and low-abundanceions at m/z 159 (loss of H2O from m/z 177), m/z 133 (loss ofC2H2 from m/z 159), m/z 151 (loss of C2H2 from m/z 177),

O

m/z 15

O

m/z 133

O NH2

m/z 150

m/z 165

H

-NH3

-C2H2

-C2H2

Scheme 7. Proposed fragmentation mechanism for DP8 (m/z 226).

Biomed. Chromatogr. 2012; 26: 720736 Copyright 2011 JohnNH2

OH

z 226

O NH2

H3CO m/z 208

-H20

O

H3CO m/z 191

-NH3

NH2

OH

4

NH2

6

H

H

H

H

NH2H O NH2

m/z 98

H-H2O

Hm/z 105 (protonated styrene), m/z 91 (C7H7+), m/z 72 (C4H10N

+)and m/z 56 (C3H6N

+; Fig. 2j, Scheme 11). These fragmentationpathways were conrmed by MSn experiments. The molecularmass of DP14 was found to correspond to the molecular massof impurity I (www.tlcpharmachem.com). The elemental compo-sitions of DP14 and its fragments ions were conrmed byaccurate mass measurements (Table 9). Based on the fragmenta-tion pattern and accurate mass measurements, the possiblestructure 1-(4-vinylphenoxy)-3-(isopropyl amino) propan-2-ol canbe 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 condi-tions, whereas DP6 formed under both acidic and oxidativestress conditions (Rt = 5.0 and 5.8min, respectively). The ESI-MSof DP5 and DP6 showed abundant [M+H]

+ ions at m/z 137and m/z 167, respectively. The mass difference between DP5and DP6 was CH2O (30Da). Based on elemental compositionsand accurate mass measurements (Table 5), DP5 and DP6 wereidentied as protonated 1-(2-methoxyethyl) benzene andprotonated 1-methoxy-4-(2 methoxyethyl) benzene, respec-tively. Similarly, DP3 (Rt 3.8; m/z 135) was identied as proton-ated 1-(2-methoxyvinyl) benzene.

9

H

H

O NH2

m/z 148

-C2H4

Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/bmc

733

z 1

R. M. Borkar et al.

734Table 9. Elemental compositions for daughter ions of DP9 (m/DP10 ([M+H]+, m/z 58) and DP12 ([M+H]

+, m/z 60). TheDP10 and DP12 formed under acidic stress conditions with Rtof 12.0 and 17.0min, respectively. Based on elemental

Degradation product Proposed formula Observed mass (Da)

DP9 C6H14NO 116.1092C3H8NO 74.0611C3H9N 59.0481C9H11O2 56.0491C3H5O 57.0336C21H39N2O4 383.2911

DP11 C18H30NO4 324.2171C18H28NO3 306.2053C12H26NO3 232.1911C15H26NO3 268.1912C6H14NO 116.1092C13H20NO2 222.1471

DP13 C10H11O2 163.0751C9H9O2 149.0591C9H11O 135.0812C5H7O2 99.0412C4H12N 74.0912C3H8N 58.0655C14H22NO2 236.1655C11H13O2 177.0911C11H11O 159.0814

DP14 C9H11O2 151.0759C9H9O 133.0632C8H9 105.0681C6H12N 98.0954C7H7 91.0511C4H10N 72.0811C3H6N 56.0491

H2CNH

OH

H3C

H3CNH

O

m/z 116

H3C

O

CH3m/z 59

H

-C3H-C3H5NH2

m/z 57H3C

OH

CH

-N

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

Copyright 2011 Johnwileyonlinelibrary.com/journal/bmc16), DP11 (m/z 383), DP13 (m/z 222) and DP14 (m/z 236)compositions and accurate mass measurements (Table 5), DP10and DP12 were identied as protonated propan-2-imine andprotonated propan-2-amine, respectively.

Calculated mass (Da) Error (ppm) Proposed neutral loss

116.1070 4.12 74.0600 1.16 C3H659.0491 3.66 C3H5NH256.0495 0.92 H2O57.0335 0.16 NH3

383.2904 3.15 324.2169 4.16 C3H7NH2306.2064 4.91 H2O232.1907 3.16 C6H5O268.1907 2.18 C6H13NO116.1070 4.12 C9H12O2222.1489 5.12 163.0754 1.26 C3H7NH2149.0597 1.91 C4H9NH2135.0804 3.12 CO99.0441 5.61 C5H474.0964 4.62 C9H8O258.0651 2.12 C1OH12O2

236.1645 3.11 CH4O177.0910 1.96 C3H9N159.0804 2.12 H2O151.0754 1.89 C2H2133.0648 2.96 C2H2105.0699 4.10 C6H13NO298.0964 2.62 C8H10O291.0542 4.22 C7H15NO272.0808 3.14 C10H12O256.0495 0.92 C11H16O2

NH2

O

m/z 74

H

H2CNH2

H

6

m/z 56

-H2OH3

H

Biomed. Chromatogr. 2012; 26: 720736Wiley & Sons, Ltd.

NO

Degradation products of metoprololH3CO

-C6H13Conclusions

Stress degradation studies on metoprolol, carried out accordingto ICH guidelines, provided information on the degradationbehavior of the metoprolol under the conditions of hydrolysisand oxidation. The liquid chromatography method describedin the present study can resolve all the degradation productsfrom the metoprolol as well as from each other under various

O

OH

H3CO m/z 268

H2N

OHHN

m/z 116

H3CO

m/z

-C9H12O2

Scheme 9. Proposed fragmentation mechanism for DP11 (m/z 383).

O NH

OH

CH3m/z 222

OCH2

OH

CH3m/z 163

H3CNH

m/z 74

m/z 58

O

H3Cm/z 135

CH2

CH

O

O

m/z 99

-C3H7NH2

-C9H8O2

-C5H4-CO

H

H2N

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

Biomed. Chromatogr. 2012; 26: 720736 Copyright 2011 JohnOHN

OH

CH2

HOHN

m/z 383

-C3H7NH2stress conditions. The drug showed extensive degradation inacid, base hydrolysis and oxidative stress, while it was stable toneutral, thermal and photolytic stress conditions. A total of 14degradation products were characterized with the help of theMS/MS, MSn experiments combined with accurate massmeasurements of fragment ions and precursors. Of these degra-dation products, DP2 (m/z 153) and DP14 (m/z 236) matchedimpurities B and I, respectively.

OHN

OH

CH2

HOH3CO m/z 324

m/z 306HN

OH

CH2

HO 232

-C6H4O -H2O

O

OH

CH3m/z 149

H

-C4H9NH2

H

CH2

H

Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/bmc

735

23

/z 1

NHH

m/z 91

R. M. Borkar et al.

736O

m

O

O

O H

m/z 151-H20

-C2H2O

m/z

O

NH

m/z 72

NH

m/z 56

-C3H9N-CH4AcknowledgmentsThe authors thank Dr J. S. Yadav, Director, IICT, Hyderabad andDr Ahmed Kamal, Project Director, NIPER, Hyderabad for facilitiesand their cooperation. B.R. is thankful to DST for the award of aJunior Research Fellowship. R.B. wishes to thank the manage-ment of the United States Pharmacopeia Laboratory, India forsupporting this work.

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m/z 15

O

m/z 133

-C2H2

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

Copyright 2011 Johnwileyonlinelibrary.com/journal/bmcOH HNH

6

OH H

O

77

CH

H

m/z 105

Hm/z 98British Pharmacopoeia, medicinal and pharmaceutical substances. 2009;I and II 39333936.

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Johnson RD and Lewis RJ. Quantitation of atenolol, metoprolol, andpropranolol in postmortem human uid and tissue specimens viaLC/APCI-MS. Forensic Science International 2006; 156: 106117.

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Yilmaz B, Asci A and Arslan S. Detection. Journal of Separation Science2010; 33(13): 19041908.

9

C H

Biomed. Chromatogr. 2012; 26: 720736Wiley & Sons, Ltd.