Journal of Pharmaceutical and Biomedical Analysis 85 (2013) 123–131
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Journal of Pharmaceutical and Biomedical Analysis
journa l homepage: www.e lsev ier .com/ locate / jpba
haracterization of degradation products of idarubicin throughC-UV, MSn and LC–MS-TOF studies
heeraj Kaushik, Gulshan Bansal ∗
epartment of Pharmaceutical Sciences and Drug Research, Punjabi University, Patiala 147002, India
r t i c l e i n f o
rticle history:eceived 16 April 2013eceived in revised form 26 June 2013ccepted 7 July 2013vailable online 24 July 2013
eywords:darubicinorced degradation
a b s t r a c t
Idarubicin was subjected to forced degradation under the ICH recommended conditions of hydrolysis,oxidation, dry heat and photolysis to characterize its possible impurities and/or degradation products.The drug was found unstable to acid hydrolysis at 85 ◦C and to alkaline hydrolysis, and oxidation at roomtemperature. The hydrolytic and oxidative degradation products were resolved with gradient and iso-cratic elution, respectively on an Inertsil RP18 (250 mm × 4.6 mm; 5 �) column with HCOONH4 (20 mM,pH 3.0) and acetonitrile. The drug degraded to two products (O-I and O-II) in oxidative condition, twoproducts (K-I and K-II) in alkaline hydrolytic, and one product (A-I) in acidic hydrolytic conditions. Thepurity of each in the LC-UV chromatogram was ascertained through LC-PDA analysis. The products were
n
C-MS-TOFegradation productass fragmentation
characterized through +ESI-MS studies on the drug and LC–MS-TOF studies on the degraded drug solu-tions. Based on the multistage mass fragmentation pattern of idarubicin and accurate mass analysis ofthe degradation products, the O-I, O-II and A-I were characterized as desacetylidarubicin hydroperox-ide, desacetylidarubicin and deglucosaminylidarubicin, respectively. The products K-I and K-II were notcharacterized due to their low concentrations and/or extremely weak ionization. The mechanisms ofdegradation of idarubicin to these products were proposed and discussed.
. Introduction
Idarubicin (IDA) (Fig. 1) is an anthracycline based antineoplasticrug used for the treatment of acute myelogenous leukemia andhronic lymphocyctic leukemia in adults [1]. It is a 4-demethoxynalog of daunorubicin and approved by US-FDA for better effi-acy and less cardiotoxicity in comparison to other anthracyclinentineoplastics. The higher activity of IDA vis-a-vis daunorubicins attributed to the absence of 4-methoxy moiety in it [2,3]. Its official in United States Pharmacopeia [4] wherein no relatedubstance or impurity is mentioned in the monograph. However,he ICH guidelines Q3A and Q3B require the characterization of allmpurities (process and degradation related) that may be presentn a drug substance or product [5,6]. This characterization is facil-tated by forced degradation of the drug under varied conditionsf hydrolysis, oxidation, dry heat, and photolysis [7]. Some analyt-cal methods for determination of IDA along with its metabolites
r in the presence of other members of this class are reported initerature [8,9]. Yang and Guo [10] have developed a HPLC methodor determination of IDA and its related substances. But there is
no report on degradation studies or degradation products of IDA.Hence, the present study has been designed to (i) conduct forceddegradation study on IDA under the ICH prescribed conditions toidentify all possible degradation products that may form under var-ious conditions like hydrolysis, photolysis, dry heat, and oxidation;(ii) characterize the degradation products through spectral and/orLC–MS-TOF studies and (iii) establish its degradation pathways andintrinsic stability characteristics. The drug has been found stableto photolytic and thermal degradation but it undergoes extensivedegradation in alkaline media. One acidic hydrolytic and two oxida-tive degradation products have been characterized and the mostprobable mechanisms of degradation are outlined and discussed.
2. Experimental
2.1. Drug and chemicals
Idarubicin hydrochloride (IDA) was procured as gift samplesfrom Strides Arcolabs Pvt. Ltd. (Bangaluru, India) and used with-out further purification. Sodium hydroxide (NaOH), hydrochloricacid (HCl), hydrogen peroxide (H2O2, 30%) and ammonium for-
mate were purchased from Loba Chemical Pvt. Ltd. (Mumbai,India). Methanol, formic acid and acetonitrile (HPLC grade) werepurchased from Merck Specialist Pvt. Ltd. (Mumbai, India). HPLCgrade water was obtained from the Direct Ultra water purification
124 D. Kaushik, G. Bansal / Journal of Pharmaceutical a
sN
2
uirNtcwwIoaiRiamGs(rLtoGvs(as
2
waaFofitwa
Fig. 1. Structure of idarubicin (IDA).
ystem in the laboratory (Bio-Age Equipment and Services, SASagar, India).
.2. Equipments
Hydrolytic and thermal forced degradations were carried outsing high precision water bath and hot air oven equipped with dig-
tal temperature control capable of controlling temperature withinange of ±1 ◦C and ±2 ◦C, respectively (Narang Scientific Works,ew Delhi, India). Photodegradation was carried out in a pho-
ostability chamber (KBF 240, WTB Binder, Tuttlingen, Germany)apable of controlling temperature and relative humidity (RH)ithin a range of ±2 ◦C and ±5% RH, respectively. The chamberas equipped with light sources as described in Option 2 in the
CH guideline Q1B [11]. The chamber was set at a temperaturef 25 ◦C and RH of 55%. The forced degradation samples werenalyzed on a Waters HPLC system (Milford, MA, USA) consist-ng of binary pumps (515), dual wavelength detector (2487) andheodyne manual injector. The data were acquired and processed
n Empower 2 software. The chromatographic separations werechieved on Inertsil RP18 (250 mm × 4.6 mm; 5 �) column. Theobile phase was degassed using ultrasonic bath (570/H ELMA,ermany). LC-PDA analysis was performed on Waters binary HPLCystem (Milford, MA, USA) consisting of pumps (515), auto injector2707), and PDA detector (2998). MSn studies on IDA were car-ied out using positive mode of electrospray ionization (+ESI) onTQ-XL ion trap quadrupole mass spectrometer (Thermo Scien-ific, Germany). LC–MS-TOF studies were carried out in +ESI moden micrOTOF-Q11 mass spectrometer (Bruker Daltonics GmbH,ermany), which was controlled by microTOF control softwareer.2.0. LC part of the LC–MS comprised of Agilent 1100 series LCystem (Agilent Technologies Inc, CA, USA) controlled by HystarVer. 3.1) software. A splitter was placed before the mass detector,llowing entry of only 35% of the eluent. Column used for LC–MStudy was same as that for LC-UV study.
.3. Forced degradation study
The hydrolytic degradation studies on IDA were carried out inater, 0.1 M NaOH and 0.1 M, 1 M and 2 M HCl at 80 ◦C for 8 h. The
lkaline hydrolytic degradation study was also carried out in 0.1 Mnd 0.01 M NaOH at 80 ◦C for 4 h and in 0.01 M NaOH at 40 ◦C for 2 h.or oxidative degradation, about 0.1 g of IDA dispersed in 100 mlf 30% H2O2 was placed in dark at room temperature (30 ± 5 ◦C)or 24 h. Thermal degradation was carried out on solid drug sealed
n amber color vials by exposure to 50 ◦C for 30 days. The pho-olytic degradation study was carried out on the drug in solid asell as solution state. For solution state photolytic studies, 2 ml of0.1% (w/v) solution of IDA in acetonitrile was mixed with 3 ml
nd Biomedical Analysis 85 (2013) 123–131
of each stressor separately (i.e., 0.1 M HCl, 0.1 M NaOH and H2O)in transparent glass vials. For solid state photolytic study, the drugwas spread as a thin layer in petri-plates. These vials as well as thepetri-plates were placed at a distance of 9′′ from the light sourcein the photolytic chamber for 14 days during which the total UVand white light exposure equaled about 200 Wh m−2 and 1.2 mil-lion lux h, respectively. A parallel set of same vials and petri-plateswas kept in dark under similar conditions of temperature and RHfor the same period of time to serve as dark control. Each degradedsample was refrigerated till analysis.
2.4. LC-UV method and sample preparation
The drug and its UV active degradation products were resolvedon Inertsil RP18 (250 mm x 4.6 mm; 5 �) column at ambienttemperature (30 ◦C) using 254 nm as detection wavelength. Thehydrolytic degradation products were resolved with mobile phaseA (HCOONH4, 20 mM, pH 3.0) and mobile phase B (acetonitrile)in gradient mode (0–20 min; A 90%, B 10% → 21–21 min; A 60%, B40% → 21–80 min; A 60%, B 40% → 80–81 min; A 90%, B 10%). Theoxidative degradation products were resolved through isocraticelution with mobile phase A and mobile phase B (70:30, v/v). Inboth the methods, the mobile phase flow rate was 0.7 ml min−1
and the injection volume was fixed at 20 �l. The solid drug samplesfrom thermal and photolytic conditions were rendered into solu-tions (1 mg ml−1) in methanol and were analyzed using the gradientelution method. Each degraded drug solution as well as solution ofsolid drug samples was diluted up to 10 times with mobile phaseA. The acid and alkali hydrolyzed solutions were neutralized beforedilution. Each dilute sample was filtered through nylon membrane(0.45 �) before analysis. The LC-UV analysis of each sample was pre-ceded by the corresponding blank. The LC-PDA studies were carriedout to check the purity of IDA and each degradation product peakresolved in the LC-UV chromatograms.
2.5. +ESI-MSn and LC–MS-TOF studies
Five stage mass (MS5) spectra of IDA were recorded in +ESImode using appropriately chosen precursor ions and ionizationpotentials (18.0–28.0 V). The operating conditions for recording MSscan of IDA were optimized as follows; end plate offset voltage,−500 V; capillary voltage 4500 V; collision cell RF, 400.0 vpp nebu-lizer, 1.2 bar; dry gas, 6.0 l min−1 and dry temperature, 200 ◦C. Thesame operating conditions were employed for recording mass spec-tra of IDA and degradation products at the ionization potentials of10 and 15 V during LC–MS-TOF analysis. All MS5 and LC–MS-TOFspectra were recorded in the range of 50–1000 m/z.
3. Results and discussion
3.1. LC-UV method
A HPLC method for determination of IDA as reported by Wallet al. [12] was taken as a lead to develop a method for separa-tion of IDA and its UV active degradation products in a singlerun. After numerous experimental trials, IDA and the degra-dation products were optimally resolved on an Inertsil RP18(250 mm × 4.6 mm; 5 �) column using gradient elution with mobilephase A (water:acetonitrile, 90:10, pH 2.5) and mobile phase B(methanol: water, 55:45, pH 2.5) at a flow rate of 1 ml min−1. Whilethe alkaline degradation products were resolved with mobile phaseA, the acidic and oxidative degradation products were resolved
with mobile phase B. However, the LC–MS studies of the degrada-tion sample using these chromatographic conditions did not showany peak in the total ion chromatogram (TIC). Even an increase inionization energy did not produce any peak in TIC. It was attributed
D. Kaushik, G. Bansal / Journal of Pharmaceutical and Biomedical Analysis 85 (2013) 123–131 125
ution
tirAwweitBt8twdsIrtd
3
cottde(8(a
as well as in dry heat and photolytic conditions. The LC-PDA anal-ysis of the degradation samples revealed that purity angles of thepeaks due to IDA, A-I, O-I, O-II, K-I and K-II were less than their
Table 1Peak purity data.
Analyte peak Purity angle Purity threshold
IDA 0.784 2.538A-I 0.629 1.693
Fig. 2. Chromatogram of standard solution of IDA (A), of IDA sol
o the absence of ionization facilitators (such as inorganic salts)n the mobile phase. Hence, water in both the mobile phases waseplaced with ammonium acetate or ammonium formate buffer.mmonium acetate buffer of different strengths (10, 20 and 50 mM)ith varied pH (2.5–5) did not resolve the degradation producthereas ammonium formate buffer (20 mM, pH 2.5) afforded mod-
rate resolution. Further replacement of methanol with acetonitrilen mobile phase B improved the elution and resolution of the oxida-ive products. The best resolution was obtained with mobile phasecomposed of ammonium formate buffer (20 mM, pH 3) and ace-
onitrile (60:40, v/v) but the run time required was more than0 min. In order to shorten the run time, acetonitrile was increasedo 60% which separated the acid hydrolyzed product from the drugithin 60 min but the oxidation products were merged with therug peak. Hence, these gradient chromatographic conditions wereelected for separation of alkali and acid degradation products ofDA whereas a separate isocratic HPLC method was developed toesolve the oxidative degradation products as disclosed in Sec-ion 2.4. The LC-UV chromatograms of standard, hydrolyticallyegraded and oxidized IDA are given in Fig. 2.
.2. Forced degradation behavior
IDA was detected as a sharp peak at 24–26 min in both the iso-ratic as well as gradient methods (Fig. 2A). LC-UV chromatogramsf the IDA standard solution as well as of each degraded solu-ion was compared with those of the corresponding and similarlyreated blank to locate the peaks due to degradation products. Therug degraded significantly to two products (O-I and O-II) afterxposure to oxidative stress at room temperature in dark for 24 h
Fig. 2B). It was extensively degraded in 0.1 M NaOH at 80 ◦C afterh. Exposure of the drug to mild alkaline hydrolytic conditions
i.e., 0.01 M NaOH; degradation temperature, 40 ◦C or ambient;nd degradation time, 4 h or 30 min) produced the same pattern
s subjected to 30% H2O2 (B) and to 0.1 M NaOH and 2 M HCl (C).
of degradation impurities which was hard to resolve. Despite theextent and pattern of drug degradation being independent of sever-ity of the alkaline hydrolytic conditions, the UV absorption spectraof variedly alkali degraded drug solutions were found to be simi-lar to that of the standard drug solution. It suggested that thoughthe drug is highly susceptible to hydrolysis in alkaline medium,the degradation was not accompanied with any change in chro-mophore of the drug. The drug was stable when exposed to 0.1 MHCl at 80 ◦C for 8 h. Increase in acid strength to 1 M resulted ininsignificant degradation with formation of a single product (A-1)as a trace peak. However, further increase in acid strength to 2 Msignificantly degraded the drug to A-1 after 8 h. It suggested thatthough the acid strength did not affect the pattern of drug degrada-tion but it severely affected the extent of degradation. The two alkalihydrolyzed products (K-I and K-II) and the single acid hydrolyzedproduct (A-1) were optimally resolved in a single run (Fig. 2C). Nodegradation was seen in water at 80 ◦C for 8 h, thermal stress for 30days, and the photolytic stress. Based on these results, the drug hasbeen found to be extremely unstable in alkaline medium, unstablein oxidative and strong acidic media but stable in neutral medium
126 D. Kaushik, G. Bansal / Journal of Pharmaceutical and Biomedical Analysis 85 (2013) 123–131
Fig. 3. Five stage mass fragmentation spectra of IDA.
D. Kaushik, G. Bansal / Journal of Pharmaceutical and Biomedical Analysis 85 (2013) 123–131 127
ttern o
pp
3
aso(m(Ta3a
333 in MS3 Spectrum (Fig. 3C). The m/z 462 fragmented to m/z 444and 388 in MS4 spectrum (Fig. 3D) and the m/z 436 fragmented tosingle fragment of m/z 291 in MS5 spectrum (Fig. 3E). Based on this
Table 2Precursor (parent) ions and product (daughter) ions in MS5 studies.
MSn stage Precursor ion (m/z) Product ions (m/z)
MS1 – 498 (Parent ion) (100%)
Fig. 4. Proposed mass fragmentation pa
urity thresholds (Table 1). It suggested that all these peaks wereure and no other product co-eluted with these peaks.
.3. Mass fragmentation pattern of IDA
Some reports on mass spectrometric analysis of IDA are avail-ble in literature [13–15] but there is no study on multistage masspectrometric analysis and hence no mass fragmentation patternf the drug is known so far. In the present study, five stage massMS5) spectra of IDA were recorded (Fig. 3) to outline its mass frag-
entation pattern. The drug was detected at m/z 498 as [M+H+] ionparent ion) corresponding to its molecular mass of 497 Da (Fig. 3A).
he various product ions formed from different precursors ions inll five stages are summarized in Table 2. The product ions m/z51, 333, 291 and 148 have also been detected in CI-MS, CID-MSnd +ESI-MS spectra of idarubicin as reported by Beijnen et al. [13],
f the IDA and its degradation products.
Sleno et al. [14] and Lachatre et al. [15], respectively. A critical studyof the MS5 spectra revealed that the parent ion directly fragmentedto m/z 480, 454, 369, 351, 148 in MS2 spectrum (Fig. 3B). The heavi-est fragment m/z 480 further fragmented to m/z 462, 436, 368 and
128 D. Kaushik, G. Bansal / Journal of Pharmaceutical and Biomedical Analysis 85 (2013) 123–131
, ‘**’ an
dmatfodw4Cs4ttoa
Fig. 5. LC–MS-TOF spectra of IDA and its degradation products. The ‘*’
ata, mass fragmentation pattern of IDA was outlined (Fig. 4). Frag-entation of the parent ion to m/z 480 and 454 by the loss of 18 Da
nd 44 Da was attributed to the loss of H2O and CH3CHO, respec-ively. The fragments m/z 369 and 351 were proposed to form byragmentation of the glucosamine moiety, similarly as fragmentsf m/z 415 and 397 have been reported to form from parent ion ofoxorubicin [14]. Taking a clue from the same report, the m/z 148as assigned to the glucosamine ion. In MS3 spectrum, the m/z 462,
36, 368 and 333 were proposed to form by loss of a water molecule,H3CHO molecule and by fragmentation of the glucosamine moietyimilarly as in MS2 spectrum. In MS4, the m/z 462 fragmented to m/z44 by the loss of a H2O molecule and to m/z 388 due to fragmen-
ation of the glucosamine chain. In MS5, the m/z 436 fragmented tohe single product ion of m/z 291 which was possible due to the lossf glucosamine moiety. In addition to the MS5 study, the drug waslso analyzed through LC–MS-TOF to generate an accurate mass
d ‘***’ represent the parent, M+Na+ and M+K+ ion peaks, respectively.
spectral data. The TOF spectrum of the drug (Fig. 5A) showed par-ent ion at m/z 498.1748 in addition to Na+ and K+ adduct ions at m/z520.1566 (M+Na+), m/z 536.1300 (M+K+), respectively. The majorfragments were noted at m/z 351.0850, 333.0735, 291.0632. Thesemasses were found to match very closely with accurate masses ofthe fragments of m/z 351, 333 and 291 (Fig. 4) which supported theproposed structural assignments to these ions.
3.4. Characterization of degradation products
The hydrolyzed and oxidized drug solutions were analyzed onLC–MS-TOF instrument to characterize the degradation products
through the mass spectral data. The two oxidative degradationproducts (O-I and O-II) and the single acid hydrolyzed product (A-I)were detected in TIC similarly as in LC-UV chromatogram. How-ever, the alkali degraded products (K-I and K-II) were not detected
D. Kaushik, G. Bansal / Journal of Pharmaceutical and Biomedical Analysis 85 (2013) 123–131 129
Table 3LC–MS-TOF spectral data of the idarubicin and its degradation products.
Analyte
peak
Observed
Mass (Da)
Mass
difference
Most probable composition (Theoretical mass; Tolerance)a
n TIC. These observations in comparison to the results obtainedith LC–MS analysis using buffer free mobile phase (as disclosed
n Section 3.1) indicated that ionization of these products was notffected by the presence or absence of ionization facilitator in theobile phase. Even the use of varied ionization potentials did not
elp in ionization of K-I and K-II. Hence, these products were sug-ested to remain absent in TIC due to their low concentrationsnd/or weak inherent ionization. The MS-TOF spectra of O-I, O-IInd A-I are given in Fig. 5. The most probable molecular formula
corresponding to each parent and fragment ion was calculated byElemental Composition Calculator Software and given in Table 3.The tolerance between the observed and calculated accuratemasses of each ion was set at not beyond ±5%.
3.4.1. Product O-IIIt was detected as cluster of peaks at m/z 454.1502, 476.1326
and 492.1071 (Fig. 5C). Based on the mass differences among thesepeaks, these were assigned respectively, as the parent ion (M1),
130 D. Kaushik, G. Bansal / Journal of Pharmaceutical and Biomedical Analysis 85 (2013) 123–131
ation
MapIfwbpmOb3
3
wrpg[p1dnhtadHoOuTl(pwp
Fig. 6. Proposed mechanism of form
+Na+, and M+K+ adduct ions of O-II. Incidentally, the M1 waslso noted as fragment ion in MS2 spectrum of IDA wherein it wasroposed to form by the loss of CH3CHO from the parent ion of
DA. Moreover, the accurate mass of this fragment in MS2 wasound to match very closely with that of M1 (Fig. 4). Hence, O-IIas proposed to be desacetylidarubicin which might be formed
y oxidative deacetylation through Baeyer Villiger oxidation in theresence of H2O2 [16]. This proposition was supported by frag-ents of m/z 325.0716 and 307.0596 in LC–MS-TOF spectrum of-II which were formed from the proposed desacetylidarubiciny the loss of glucosamine moiety similarly as m/z 351.0850 and33.0735 were formed from the parent drug ion (Fig. 4).
.4.2. Product O-IIt was recorded as the major heavy ion at m/z 488.1563 along
ith its M+Na+ and M+K+ ions at m/z 510.1374 and m/z 526.1122,espectively (Fig. 5B). Hence m/z 488.1563 was assigned as thearent ion (M2) of O-I. An even molecular mass of M2 peak sug-ested an odd number of nitrogen atom (reverse nitrogen rule)17]. Hence the single nitrogen atom present in glucosamine com-onent of the IDA was suggested to be intact in O-I. The M2 was0.0185 Da less than the parent ion of IDA and this mass differenceid not correspond to any neutral molecule. It indicated that O-I wasot formed directly from the drug. However, M2 was 34.0048 Daeavier than M1 and this mass difference was found to correspondo a H2O2 molecule (34.0055 Da) [18]. Further, M1 was also noteds a fragment ion in MS-TOF spectrum of O-I. Hence, based on thisiscussion, O-I was suggested to form from O-II by the addition of2O2. This addition is possible across the ketone group generatedn the ring D in O-II to form hydroxyhydroperoxide [19]. Hence,-I was proposed to be desacetylidarubicin hydroperoxide whichndergoes mass fragmentation (Fig. 4) in consonant with its MS-OF spectrum. The heaviest fragment of m/z 470.1470 (18.0093 Daess than M2) was possible to form by the loss of a water molecule
18.0105 Da) whereas the fragment m/z 454.1515 (O-II) was pro-osed to form by the loss of H2O2 molecule. Fragment m/z 363.0474as 147.0900 Da less than the M+Na+ ion of O-I. Hence, it was pro-osed to form by the loss of glucosamine from the M+Na+ ion of
of degradation products from IDA.
O-I. The fragment m/z 341.0654 was possible to form by loss ofglucosamine moiety from M2 similar to the fragmentation of theparent drug ion to m/z 351.0850. The fragments of m/z 325.0695 and307.0593 were possible to form from M1 as discussed in Section3.4.1.
3.4.3. Product A-IIt was detected as a minor peak at m/z 369.0970, a major
ion peak at m/z 391.0790 along with heaviest ion peak at m/z407.0545 (Fig. 5C). Based on the mass differences amongst thesepeaks, the m/z 369.0970, 391.0790 and 407.0545 were assignedas parent ion (M3), M+Na+ and M+K+ ions, respectively. An oddmolecular mass of M3 suggested an even number or no nitrogenatoms (reverse nitrogen rule) [17]. Therefore, the single nitro-gen atom in the glucosamine component of IDA was expected tobe lost during degradation of IDA to A-I. The m/z 369.0970 was129.0778 Da less than the parent ion of IDA and this mass loss cor-responded to the glucosamine moiety. Hence, A-I was proposed tobe de-glucosaminyl idarubicin which could be formed by cleavageof glycosidic linkage between the tetracycline ring and the glu-cosamine moiety. This proposition was supported by the fact thatM3 was also noted as fragment in MS2 spectra of IDA (Fig. 3). Hence,based on this MS-TOF and MS2 spectral data, A-1 was characterizedas deglucosaminylidarubicin, which undergo mass fragmentation(Fig. 4) in agreement with its LC–MS-TOF spectrum. The prod-uct ions of m/z 351.0868, m/z 333.0761 and m/z 291.0656 (Fig. 5)were formed from M3 due to the loss of a water molecule, twowater molecules and two water molecules along with acetyl group,respectively. The m/z 373.0697 and m/z 355.0580 were proposedto be the Na+ adduct ion of fragments 351.0868 and m/z 333.0761,respectively.
3.5. Drug degradation mechanisms
The most probable mechanisms of formation of O-I, O-II and A-Iare outlined in Fig. 6. The O-II (desacetylidarubicin) was proposedto form by oxidative deacetylation of IDA through Baeyer Villigeroxidation in the presence of H2O2. The mechanistic explanation for
tical a
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D. Kaushik, G. Bansal / Journal of Pharmaceu
his oxidation is as follows [16,19]: Addition of H2O2 to carbonylond of the acetyl group on ring D forms a hydroperoxide inter-ediate which undergoes alkyl group migration. The secondary
arbon (attached to hydroxyl group), which is actually a part of ring, migrates in preference over the methyl group due to their differ-ntial migration aptitudes. It converts the acetyl group to acetateroup, which is actually a part of hemiacetal. The ester is readilyydrolyzed in the aqueous medium to generate an extremelynstable hemiacetal intermediate. The latter is immediately con-erted to O-II as ketone. However, the ketonic O-II may undergoeto-enol tautomerism to form O-II as an enol. Hence, O-II can existn both keto and enol form. Formation of O-I from O-II was pos-ible through the well known addition of H2O2 to ketone group,enerated on the ring D in O-II, to form a hydroxyhydroperoxide19]. However, this addition reaction is reversible and the hydrox-hydroperoxides (O-I) decomposes to generate the reactant (O-II).ased on this discussion it is proposed that O-I and O-II exist inquilibrium. The A-I, characterized as deglycosylidarubicin, wasossible to form by the well known acid catalyzed hydrolysis of gly-osidic linkage. Similar type of glycosidic cleavage is also reportedo occur in other anthracycline antineoplastics such as doxorubicin,′-deoxydoxorubicin, 4′-O-methyldoxorubicin, 4′-epidoxorubicin,oxorubininol, daunorubicin and carminomycin under acidic con-itions [13]. However, A-I was detected as a minute peak in drugolution hydrolyzed with 1 M HCl but it was the major peak whenhe acid strength was increased to 2 M. This difference in the ratef deglycosylation of IDA was attributed to the fact that acid-atalysed hydrolysis of glycosides bearing an aglycon moiety inxial orientation is much slower than that of glycosides bearingn aglycon moiety in equitorial orientation [20]. A 3D energy min-mized structure of IDA has revealed that the glycone part exist inhair conformation and the aglycone part (anthrquinone ring) isxially oriented with respect to the glycone part. Further, the ter-iary alcohol (geminal to the acetyl group) in ring D of IDA wasxpected to undergo dehydration readily in acid medium. But nocid hydrolyzed product with mass 18 Da less than the IDA or A-Ias detected in the TIC which indicated that this tertiary alcoholas stable in the acid medium. This exceptional stability of the ter-
iary alcohol may be attributed to the intramolecular H-bondingetween the geminal hydroxy and carbonyl groups in ring D which
n turn decreases nucleophilicity of the alcoholic oxygen makingt significantly less susceptible to protonation in the acid mediumnd hence stable to dehydration.
. Conclusion
Forced degradation studies on idarubicin were conducted underhe ICH prescribed conditions. The drug was found extremelynstable in alkaline medium, susceptible to oxidation and acidicydrolysis whereas stable to thermal and photolytic stress condi-ions. Separate LC-UV methods were developed for separation ofxidative and hydrolytic degradation products. MSn and LC–MS-OF studies were carried out to characterize the major degradationroducts. Two oxidative degradation products were character-
zed as desacetylidrrubicin and desacetylidarubicin hydroperoxide,hereas the single acid degraded product was characterized
s deglucosaminylidarudicin. The most probable mechanisms ofegradation of idarubicin were outlined and discussed.
[
[
nd Biomedical Analysis 85 (2013) 123–131 131
Acknowledgements
The authors are thankful to Strides Arcolabs Pvt. Ltd. (Bangalore,India) for providing IDA as generous gift sample and to Prof. Saran-jit Singh, Head, Department of Pharmaceutical Analysis, NationalInstitute of Pharmaceutical Education and Research (SAS Nagar,India) for extending the facilities to carry out photostability andmass spectral studies.
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