DFT for ritonavir.pdf

8/9/2019 DFT for ritonavir.pdf

1/16

Explanation through density functional theory

of the unanticipated loss of CO2 and differencesin mass fragmentation proles of ritonavir andits rCYP3A4-mediated metabolites

Shalu Jhajra,a Tarun Handa,a Sonam Bhatia,b P. V. Bharatamb andSaranjit Singha*

In the present study, the metabolism of ritonavir was explored in the presence of rCYP3A4 using a well-established strategyinvolving liquid chromatography–mass spectrometry (LC–MS) tools. A total of six metabolites were formed, of which two were

new, not reported earlier as CYP3A4-mediated metabolites. During LC–

MS studies, ritonavir was found to fragment throughsix principal pathways, many of which involved neutral loss of CO2, as indicated through 44-Da difference between massesof the precursors and the product ions. This was unusual as the drug and the precursors were devoid of a terminal carboxylicacid group. Apart from the neutral loss of CO2, marked differences were also observed among the fragmentation pathways of the drug and its metabolites having intact N -methyl moiety as compared to those lacking N -methyl moiety. These unusualfragmentation behaviours were successfully explained through energy distribution proles by application of the densityfunctional theory. Copyright © 2014 John Wiley & Sons, Ltd.

Additional supporting information may be found in the online version of this article at the publisher ’ s web site.

Keywords: ritonavir; metabolism; rCYP3A4; mass fragmentation; CO2 neutral loss; group substitution effect; density functional theory

Introduction

Mass spectrometry is an indispensable tool for drug metaboliteidentication (Met-ID) studies. In particular, high-resolution(HRMS), multi-stage (MSn) and hydrogen deuterium exchange-mass spectrometry (HDX–MS) investigations on the drug andits biotransformation products help to obtain comprehensiveinformation on the structures of the latter. A strategy for Met-ID,involving establishment of fragmentation proles of the parentand its metabolites, and their comparison, has been proposedearlier by us.[1–3] The metabolites, being similar to the parent drug,usually follow the same or parallel mass fragmentation prole, withsome of the resulting product ions differing in mass in accordancewith the changes in structural motifs where biotransformation hadtaken place. A critical comparison among various product ions

yields vital information regarding the type and site of themetabolicchange(s) involved. For example, an increase in the mass of ametabolite by 16Da owing to metabolic oxidation would usually leadto a constant mass difference of 16Da during fragmentation in all thesteps where an altered moiety remains intact. The fragments originat-ingfrom the unaltered part of the structural framework generallytendto yield product ions identical to those of the parent.

The purpose of the present study wasto investigate rCYP3A4-me-diated metabolism of ritonavir (1,3-thiazol-5-ylmethyl-N-[(2S,3S,5S)-3-hydroxy-5-[(2S)-3-methyl-2-{[methyl({[2-(propan-2-yl)-1,3-thiazol-4-yl]methyl})carbamoyl]amino}butanamido]-1,6-diphenylhexan-2-yl]carbamate, Fig. 1). We employed commercial recombinanthumanized CYP3A4 (rCYP3A4) isozyme and detected a total of six metabolites. Incidentally, a near overlapping study was reported

lately by Lin et al.,[4] in which only four (C -hydroxylated ritonavir, N -dealkylated ritonavir, N -demethylated and deacylated ritonavir) outof six metabolites were detected when metabolic reaction was carriedout using CYP3A4 puried from expressed Escherichia coli TOPP3 cells.

While carrying out metabolite characterization using massspectrometry tools,[2] we conducted investigations in positiveelectrospray ionization (+ESI) mode. We found that ritonavirwas dissociated through six principal mass fragmentation routes,wherein neutral loss of 44 Da was observed in many fragmenta-tion steps. The exact mass of the losses, calculated from thedifference between accurate (observed) masses of the precursorand the product ions involved, matched best to CO2(43.9898 Da). The same observation was made even withmetabolites, where loss of 44 Da occurred during MS2, MS3 orMS4 transitions. This observation was unusual, as the parent drug

and its metabolites were devoid of a terminal carboxylic acidgroup. Apart from the neutral loss of CO2, marked differenceswere even observed in fragmentation pathways of the drugand its metabolites, particularly those that had intact N -methyl

* Correspondence to: Saranjit Singh, Department of Pharmaceutical Analysis,

National Institute of Pharmaceutical Education and Research (NIPER), Sector

67, S.A.S. Nagar 160 062, Punjab, India. E-mail: [email protected]

a Department of Pharmaceutical Analysis, National Institute of Pharmaceutical Education and Research (NIPER), S.A.S. Nagar, Punjab, India

b Department of Medicinal Chemistry, National Institute of Pharmaceutical Education and Research (NIPER), S.A.S. Nagar, Punjab, India

J. Mass Spectrom. 2014, 49, 452–467 Copyright © 2014 John Wiley & Sons, Ltd.

Research article

Recei ved: 4 December 2013 Revised: 18 February 2014 Accepted: 10 March 2014 P ub lished on lin e i n Wiley On line Library

(wileyonlinelibrary.com) DOI 10.1002/jms.3359


2/16

moiety in contrast to N -dealkylated metabolites. To explain boththese odd observations, in silico energy distribution proles wereestablished by applying density functional theory (DFT).

Experimental

Chemicals and materials

Pure ritonavir was obtained as a gratis sample from RanbaxyPharmaceuticals Ltd. (Gurgaon, India). Nicotinamide adenine dinu-cleotide phosphate-reduced tetrasodium salt (NADPH), testoster-one, dipotassium hydrogen phosphate and potassium dihydrogenphosphate were purchased from HIMEDIA Laboratories Pvt. Ltd.(Mumbai, India). rCYP3A4 was acquired from BD Gentest™, BDBiosciences (California, USA). A Zorbax Eclipse XDB, C18 column(250mm×4.6mm, 5μ) from Agilent Technologies (Wilmington,DE, USA) was used for the separation of the drug and its metabolites.For liquid chromatography–mass spectrometry (LC–MS) analyses,high performance liquid chromatography grade acetonitrile (ACN)from J.T. Baker (Mexico, USA), purchased locally from RFCL Limited,Mohali, India was used. Ultra pure water was obtained from a water

purication unit (ELGA, Bucks, England).

Instruments

For liquid chromatography–high resolution mass spectrometry(LC–HRMS) studies, a 1100 series LC from Agilent Technologies(Waldronn, Germany) connected to a time-of-ight MS (MicrOTOF-Q, Bruker Daltonics, Bremen, Germany) was used. The entire unitwas operated using Hystar (ver. 3.1) and MicrOTOF control (ver. 2.0)software. Liquid chromatography–multiple stage mass spectrometry(LC–MSn) data were acquired using Accela™ LC (Thermo ElectronCorporation, SanJose, USA), which was connected to a linear ion trapquadrupole massspectrometer (LTQ–XL™, ThermoElectron Corpora-

tion). Instrument control and data collection were carried out byusing Xcalibur software (ver. 2.0.7 SP1). The mass studies were essen-tially carried out in +ESI mode after optimization of mass operationparameters. A 5-mM solution of sodium formate (Sigma-AldrichChemicals, Bangalore, India) was used as a calibrant in HRMS studies.HDX–MS studies were conducted on drug using the MS/TOF system.

Methodology

In silico metabolite prediction

Before initiating experimental studies, molecular masses of CYP3A4-mediated metabolites were predicted using MetaSitesoftware (Molecular Discovery, Pinner, Middlesex, UK). This was

followed by the determination of exact (theoretical) masses of their corresponding protonated species.

In vitro metabolite generation

rCYP3A4 (nal conc. 10ppm) was added to the culture tubescontaining 40-μl phosphate buffer (100mM, pH 7.4). This mixturewas pre-incubated for 5 min at 37 ± 1 °C, followed by addition of

ritonavir (

nal conc. 10μM). Subsequently, NADPH solution (a cofactor,nal conc. 1.3 mM) was added to initiate the metabolic reaction.The volume was made up to 200μl with 100mM phosphate buffer.From each of the reaction mixtures, 20-μl aliquot was collected at0 min and quenched with an equal volume of chilled ACN. Theremaining sample was incubated at 37 ± 1 °C for 1 h for metabolicreactions to occur. Two other sets, a control devoid of NADPHand a blank without rCYP3A4, were also prepared. These solutionswere simultaneously subjected to incubation at 37 ± 1 °C for 1 h.

Mass fragmentation studies on ritonavir

For HRMS studies on the drug, a 2 μg/ml solution was directlyinjected in the MS/TOF system at a ow rate of 6 μl/min, usingan integrated syringe pump. The MSn studies were similarlyconducted by directly injecting a 5 μg/ml solution of the druginto the LTQ–XL system at a ow rate of 3 μl/min. The HDX–MSstudy was carried out by preparing drug solution in D2O:ACN(50:50) and directly injecting it into the MS/TOF system. CriticalHRMS and MSn parameters (Supplementary Table 1) wereoptimized in all these experiments, and the same were used forall subsequent LC–MS studies on the metabolites.

LC –MS (LC –HRMS and LC –MS n

) studies on the metabolites

An LC method was rst developed for the separation of the metab-olites and their characterization by mass tools. To achieve optimalseparation of the analytes from the polar matrix components, a gra-dient mode was employed, using a mobile phase composed of ACN

and 10 mM ammonium acetate (pH 4.7). The ow rate and columntemperature were 0.4ml/min and 25 °C, respectively. The initial mo-bile phase ratio of ACN:buffer was 10:90, which was maintained forrst 5 min and changed to 90:10 in 40 min. It was held at this com-position till 45 min, again brought back to initial ratio in 48 minand was equilibrated for the next 12 min. This gradient LC methodwas employed further in LC–HRMS and LC–MSn studies, which wereconducted on prepared in vitro incubated samples. Positive ioniza-tion mode was selected for LC-MS studies on the metabolites.The metabolites were not detected in negative ionization mode,probably due to lack of suitable functional groups. The parent itself also had very low ionization in the negative mode.

Strategy used for structure elucidation of the metabolites

A generic strategy for the characterization of the metabolites, pro-posed earlier from our laboratory,[2] was followed in the presentstudy. The initial step involved the generation of total ion chro-matogram (TIC) on rCYP3A4-treated drug sample (including zeromin sample) using LC–MS/TOF. It was followed by prediction of CYP3A4-mediated metabolites by stand-alone computational tool,like MetaSite. The exact mass of each predicted metabolite and of those previously reported in the literature were used to generatepost-acquisition extracted ion chromatograms (EICs). The metabo-lites present in the TIC were identied based on EICs, while thepeaks due to matrix components were segregated by comparisonof theTIC of thesample with theblank (0-min sample). Additionally,analyses of accurate mass data and monitoring of the characteristic

Figure 1. Chemical structure of ritonavir. The structure is divided into 3parts (A, B and C) for easy understanding of drug fragmentation patternand metabolic changes.

Explanation for unanticipated loss of CO2

J. Mass Spectrom. 2014, 49, 452–467 Copyright © 2014 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jms


3/16

common fragments producedfrom the drug and the metabolites[1]

helped in identication of the latter in the samples.The accurate mass data helped in calculation of the difference in

observed mass and thus elemental composition of the metaboliteswith respect to the drug. This allowed determination of ring plusdouble bonds (RDBs), elemental composition and metabolicchange in each metabolite, including metabolic events involved.Finally, help was taken of MSn data, and the fragmentationpathways generated therefrom were employed to justify theproposed structure of each metabolite.

DFT calculations

Geometry optimizations without symmetry constraints wereperformed on the model structure of ritonavir and its metabolites(by taking relevant parts of the structures). In the case of metabo-lites, the portions where metabolic changes resulted into differentialmass fragmentation pattern were selected as the model structures.DFT calculations[5]were carried out using the B3LYP method[6,7] with6-31+ G(d) basis set as implemented in Gaussian 09 suit of packages.[8] Frequencies were computed analytically at B3LYP/

6-31+ G(d) level for all the optimized species to characterize eachstationary point as a minimum or a transition state, and to estimatethe zero-point energies (ZPE). The calculated ZPE values (at 298.15 K)were scaled by factors of 0.9806 for B3LYP level calculations.[9]

Further, the partial atomic charges on various atomic centres wereestimated by natural bond orbital analysis[10,11] using the internalmodule of Gaussian 09 at the B3LYP/6-31 + G(d) level of theory.All the transition states were characterized by single negativeimaginary frequency.

Results and discussion

Mass fragmentation pattern of ritonavir

Figure 2 shows the MS/TOF line spectrum of ritonavir in ESI positivemode. Evidently, a total of 17 fragments (as labelled in Fig. 2) were

formedfrom themolecular ion [M+ H]+. Among them, thefragmentsof m/z 426 and 296 were most abundant. Some of the fragments wereseen in MSn studies only. The most probable molecular formula for theions observed in HRMS studies was calculated from theiraccurate masses with the help of an elemental compositioncalculator (available from; http://www.wsearch.com.au/Tools/ elemental_composition_calculator.htm). Additionally, HDX–MSstudies provided information about the number of labilehydrogen atoms present in their structures. The whole data arecompiled in Table 1, which also includes HDX–MS values, accuratem/z values for the losses and their probable molecular formulae.The MSn data, which helped in establishing link among variousobserved ions, are listed separately in Table 2. A comprehensivefragmentation pattern of ritonavir was delineated taking intoaccount all available data. The same is shown in Fig. 3. Suchcomprehensive mass fragmentation of ritonavir is not previouslyreported in the literature.

Apparently, the molecular ion of ritonavir ([M+ H]+ = m/z 721)underwent fragmentation through six principal routes to form bothdifferent and common product ions (Fig. 3), highlighting theprotonation at different positions in the drug molecule. Critical

comparison among these routes also revealed that many stepswere essentially similar to each other, with some of them differingin sequence of losses. The same are individually discussed below:

Route 1: The rst route involved loss of H2O from the parent toform a product ion of m/z 703, which was attributed to theprotonation of secondary alcoholic group in the drug structure.The latter underwent further fragmentation through the path-way m/z 703→ 533→ 489→ 347, 250.

Route 2: This route involved cleavage of C30―N31 urea amide bondpresent in part C of the parent ion to form product of m/z 551,accompanied with a neutral loss of 1-(2-isopropylthiazol-4-yl)-N -methylmethanamine, due to the charge present on the tertiary

nitrogen. In this case, the product ion of m/z 533 was formed fromm/z 551 upon the loss of H2O. The latter further followed a

3 6 5

. 1 6 5 7

2 9 6

. 1 4 3 4

4 2 6

. 1 8 4 8

5 0 7

. 2 4 3 8

5 3 3

. 2 2 3 6 7

2 1

. 3 2 2 1

6 0 6

. 3 1 1 9

4 8 9

. 2 3 3 2

7 0 3

. 3 0 9 8

6 7 7

. 3 3 1 6

4 0 8

. 1 7

4 0

5 5

1 .

2 3 3 8

3 1 1

. 1 7

4 5

+MS2(721.0000), 4.2-4.5min

0.0

0.5

1.0

1.5

2.0

2.5

5x10Intens.

200 300 400 500 600 700 m/z

1 9 7

. 0 7

2 2

2 5

0 .

1 5

6 0

2 6 8

. 1 4 8 9

200

3 8 2

. 1 9 3 6

[ M + H ] +

abcdeg

h

f ik

l

m

n

o

q

r 3 4 7

. 1 5 7 7

s

Figure 2. HRMS spectrum of ritonavir in ESI positive ionization mode.

S. Jhajra et al .

wileyonlinelibrary.com/journal/jms Copyright © 2014 John Wiley & Sons, Ltd. J. Mass Spectrom. 2014, 49, 452–467

http://www.wsearch.com.au/Tools/elemental_composition_calculator.htmhttp://www.wsearch.com.au/Tools/elemental_composition_calculator.htmhttp://www.wsearch.com.au/Tools/elemental_composition_calculator.htmhttp://www.wsearch.com.au/Tools/elemental_composition_calculator.htm


4/16

pathway m/z 533→489→347, 250, same as Route 1. Along with theion of m/z 533, other fragments generated parallely from m/z 551were of m/z 507, 408, 392, and 311. Of these, the ion of m/z 507

further lost H2O to form a product of m/z 489, which subsequentlyfollowed the same sequence as in Route 1, viz., m/z 489→347, 250.

Route 3: In the third route, the parent fragmented to product ion of m/z 677, with an observed accurate mass difference of 43.9905 Da(error


5/16

(Fig. 1), attributed to charge on the carbamate oxygen. In thisfragmentation route, the product ion of m/z 606 furtherfragmented into ions of m/z 311, 296 and 268. The fragment of m/z 296 sequentially cleaved by following the pathway: m/z 296→ 268→ 197, 171.

Route 6: The sixth route involved direct fragmentation of the par-ent molecular ion to a carbonyl cation (m/z 296) equivalent topart C of the drug structure (Fig. 1). The product ion of m/z 296followed exactly the same pathway as in Route 5, where it wasformed from the precursor of m/z 606, instead of the parent.

On the whole, the unusual loss of CO2 was observed fromfragments of m/z 551, 533 and 426, leading further to ions of m/z 507, 489 and 382, respectively.

Generation of TICs of control and actual samples

A method separating the drug, its metabolites and matrix compo-nents was optimized on LC–MS/TOF by using the actual sample.The resultant TIC for both control and actual samples are shown inFig. 4.

Figure 3. Proposed mass fragmentation prole of ritonavir in ESI positive ionization mode.

32 34 36 38 40 42 44 46 Time [min]0

1000

2000

3000

4000

5000

M1 M2

M3

M4

M5

M6

lntens.

Figure 4. TIC of ritonavir metabolites after incubation with rCYP3A4. The metabolites were identied based on difference in peaks from the matrixchromatogram, which is also shown in the gure in red.

S. Jhajra et al .



6/16

Consideration of in silico predicted and previously known

metabolites

In total, 15 metabolites were predicted using MetaSite softwarefor CYP3A4-dependent metabolism of ritonavir. Additionally,the metabolites known in the literature were also taken intoaccount. The theoretical exact mass of their corresponding pro-tonated species were searched for checking their presence in

the actual samples.

Identication of metabolites in TIC of actual samples

As evident in Fig. 4, there were multiple peaks in TIC, which weredue to both the metabolites and the matrix. The comparison of TIC against the control, and consideration of other approachesoutlined in the experimental section, helped in identifying thepeaks due to the metabolites. A total of six peaks, which could beattributed to be the metabolites, are labelled in Fig. 4 as M1–M6.The consideration of EICs (Supplementary Fig. 1) for the exactmasses of predicted and previously known metabolites furtherhelped in substantiation that all these six peaks were in fact of

the metabolites. HRMS data and corresponding derived informa-tion for the metabolites are given in Table 3. As shown in Table 3,a few ions with low abundance had higher error in mmu, whichwas likely due to interference of matrix ions with them. The gener-ated MSn data are listed in Table 4.

Characterization of metabolites based on MS data

Metabolite M1

M1 was characterized as the deacylated metabolite involvingremoval of Part A from ritonavir (Fig. 1). Its Q-TOF line spectrum isshown in Supplementary Fig. 2. The structure was supportedby HRMS (Table 3) and MSn fragmentation data (Table 4). Themetabolite had accurate mass of m/z 580.3307 with a differenceof 140.9877 Da (~141) from the drug, which implied a loss of C5H3NO2S corresponding to Part A of the drug. A careful com-parison of its fragmentation pattern with the drug revealed adifference of 141 Da throughout the sequence of fragmentationobserved in the drug. For example, Route 2 in the drug viz . m/z 721→ 551→ 533 (Fig. 3) was replaced in the metabolite by thesequence m/z 580→ 410→ 392 (Fig. 5). The presence of thefragment of m/z 296 (Route 6) and m/z 285 (Route 3), similar tothe drug, pointed towards the presence of unaltered Part Band C, respectively. Moreover, considering together both thesefragments completely justied the structure of this metabolite. Forfurther conrmation, ions of m/z 296 and 285 were subjected toMS3 analysis. The former followed the same fate as in the drug by

yielding the product ions of m/z 268, 197 and 171. On the otherhand, m/z 285 cleaved to yield fragments of m/z 268 and 250, whilethe same in the case of the drug was generated in MS3 step from aminor product of m/z 677, in which case further fragmentation wasnot possible due to its lower abundance. On subsequent fragmenta-tion, the ion of m/z 285 reduced to form a product of m/z 268,which dissociated further into the ions of m/z 250 and 233. On theother hand, the ion of m/z 268, which was also formed from m/z 296, fragmented to form ions of m/z 197 and 171. This ndingestablished that despite the two fragments having the same m/

z of 268, they were actually different in structure. The fragmentof m/z 250 generated from the sequence m/z 285→ 268,indicated that Part B of the drug also remained intact duringthe biotransformation reaction.

Metabolite M2

The Q-TOF line spectrum of this metabolite is shown in Supple-mentary Fig. 2. Its HRMS and MSn fragmentation data are listedin Tables 3 and 4, respectively. This metabolite had a molecularion of m/z 582.2816, with the difference of 139.0368 Da fromthe parent drug corresponding to C7H9NS, which indicated lossfrom Part C of the drug. This was supported by the appearance

of the ion of m/z 426 (Route 4) and also by the lack of thefragment of m/z 296 (Route 6), which originated from Part Aand B, and C, respectively in the case of the drug. The MS3 andMS4 fragmentation analysis of another major fragment of m/z 525, which resulted from [M+H]+ after loss of C2H3NO, alsorevealed that the metabolism involved Part C of the drug. Thedifference of 99 Da between m/z 525 and 426 corresponded to2-amino-3-methylbutanal, a partial motif of PartC, further conrmingthat the site of metabolism was Part C only. The postulated fragmen-tation pattern of the metabolite is shown in Fig. 6.

Just like the parent drug, the loss of CO2 in this metabolite wasalso observed in the sequences: m/z 525→481 and m/z 426→382(shown in Fig. 6). These ions of m/z 525 and 426 did not have anyfree caboxlic group.

Metabolite M3

This metabolite had an accurate mass of m/z 723.2901 (Table 3),with the difference of 1.9717Da from the parent drug(m/z 721.3184). The possibility of the removal of two hydrogenatoms was eliminated, as that would have meant a differenceof 2.0156Da, almost 43.9 mDa away from the observed value.Thus an alternate possibility was considered, which involveddemethylation followed by hydroxylation, or vice versa. The onlyfacile site for demethylation existed in Part C of the structure of drug. The same was supported by HRMS (Table 3) and MSn

fragmentation data (Table 4), wherein all the fragmentscontaining Part C also had the same mass difference of

~1.9717 Da. For example, the line of m/z 296 (Route 6) seen inthe drug was replaced in the spectrum of the metabolite by lineof m/z 298, highlighting the involvement of hydroxylation anddemethylation. Due to this reason only, the fragment of m/z 298 showed a facile loss of H2O to generate a product ion of m/z 280. On other hand, the ion of m/z 426 (Route 4) thatoriginated from Parts A and B remained same as the drug. Thestructure and fragmentation pathway of this metabolite areshown in Fig. 7.

Again, similar to the parent drug, this metabolite also showedloss of CO2, which happened during conversion of ion of m/z 426to product of m/z 382 (shown in Fig. 7).

Metabolite M4

The accurate mass difference of metabolite M4 against the drugwas 15.9922 Da (~16 Da), which pointed towards its generationupon metabolic oxidation or hydroxylation of the drug. The Q-TOFline spectrum of this metabolite is shown in Supplementary Fig. 2.The corresponding HRMS and MSn data are listed in Tables 3 and4, respectively. The [M+ H]+ of the metabolite fragmented to giveions of m/z 426 and 312, as compared to m/z 426 and 296 (Route6) in the case of the drug (Fig. 3). The ion of m/z 426 (Route 4) inthe metabolite had a fragmentation pattern similar to that observedin the drug, revealing no metabolic changes in Parts A and B. Theappearance of an ion of m/z 312, with a mass of 16Da higher thanthe characteristic ion of m/z 296 in the drug, clearly indicated thatPart C of the drug was involved in the oxygenation process.




7/16

T a b l e

3 .

I n t e r p r e t a t i o n o f L C – M S / T O F d a

t a o f r i t o n a v i r m e t a b o l i t e s

M e t a - b o l i t e

L i n e N o .

M S / T O F d a t a

B e s t p o s s i b l e m o l e c u l a r

f o r m u l a

E x a c t m a s s o f t h e m o s t p r o b a b l e

s t r u c t u r e

E r r o r i n m m u

R D B

M e t a b o l i c c h a n g e

M 1

[ M + H ] +

5 8 0 . 3

3 0 7

C 3 2 H 4 6 N 5 O 3 S +

5 8 0 . 3

3 1 6

0 . 9

1 2 . 5

[ C 5 H 3 N O 2 S ]

a

4 6 3 . 2

6 8 0

C 2 7 H 3 5 N 4 O 3 +

4 6 3 . 2

7 0 4

2 . 4

1 2 . 5

b

4 1 0 . 2

4 3 8

C 2 4 H 3 2 N 3 O 3 +

4 1 0 . 2

4 3 8

0 . 0

1 0 . 5

c

3 9 2 . 2

2 7 3

C 2 4 H 3 0 N 3 O 2 +

3 9 2 . 2

3 3 3

6 . 0

1 1 . 5

d

3 7 5 . 2

0 8 9

C 2 4 H 2 7 N 2 O 2 +

3 7 5 . 2

0 6 7

2 . 2

1 2 . 5

e

3 4 7 . 2

1 3 8

C 2 3 H 2 7 N 2 O +

3 4 7 . 2

1 1 8

2 . 0

1 1 . 5

f

2 9 6 . 1

3 9 7

C 1 4

H 2 2

N 3

O 2

S +

2 9 6 . 1

4 2 7

3 . 0

5 . 5

g

2 8 5 . 1

9 2 8

C 1 8 H 2 5 N 2 O +

2 8 5 . 1

9 6 1

3 . 3

7 . 5

h

2 6 8 . 1

5 2 7

C 1 8 H 2 2 N O +

2 6 8 . 1

6 9 6

1 6 . 9

*

8 . 5

h ′

2 6 8 . 1

5 2 7

C 1 3

H 2 2

N 3

O S +

2 6 8 . 1

4 7 8

4 . 9

4 . 5

i

2 5 0 . 1

6 1 1

C 1 8 H 2 0 N +

2 5 0 . 1

5 9 0

2 . 1

9 . 5

M 2

[ M + H ] +

5 8 2 . 2

8 1 6

C 3 0 H 4 0 N 5 O 5 S +

5 8 2 . 2

7 4 5

7 . 1

1 3 . 5

[ C 7 H 9 N S ]

a

5 2 5 . 2

5 5 3

C 2 8

H 3 7

N 4

O 4

S +

5 2 5 . 2

5 3 0

2 . 3

1 2 . 5

b

5 0 7 . 2

4 7 8

C 2 8 H 3 5 N 4 O 3 S +

5 0 7 . 2

4 2 4

5 . 4

1 3 . 5

c

4 8 1 . 2

5 0 6

C 2 7 H 3 7 N 4 O 2 S +

4 8 1 . 2

6 3 2

1 2 . 6

1 1 . 5

d

4 6 3 . 2

5 4 3

C 2 7

H 3 5

N 4

O S +

4 6 3 . 2

5 2 6

1 . 7

1 2 . 5

e

4 2 6 . 1

8 6 6

C 2 3 H 2 8 N 3 O 3 S +

4 2 6 . 1

8 4 6

2 . 0

1 1 . 5

f

4 0 8 . 1

7 0 9

C 2 3 H 2 6 N 3 O 2 S +

4 0 8 . 1

7 4 0

3 . 1

1 2 . 5

g

3 8 2 . 1

8 4 7

C 2 2

H 2 8

N 3

O S +

3 8 2 . 1

9 4 8

1 0 . 1

1 0 . 5

h

3 6 5 . 1

5 7 2

C 2 2 H 2 5 N 2 O S +

3 6 5 . 1

6 8 2

1 1 . 0

1 1 . 5

i

3 4 7 . 1

4 2 8

C 2 2 H 2 3 N 2 S +

3 4 7 . 1

5 7 6

1 4 . 8

1 2 . 5

j

3 1 1 . 1

6 7 8

C 1 9 H 2 3 N 2 O 2 +

3 1 1 . 1

7 5 4

7 . 7

9 . 5

k

2 8 5 . 1

8 5 1

C 1 8 H 2 5 N 2 O +

2 8 5 . 1

9 6 1

1 1 . 0

7 . 5

l

2 6 8 . 1

6 6 4

C 1 8 H 2 2 N O +

2 6 8 . 1

6 9 6

3 . 2

8 . 5

m

2 5 0 . 1

6 1 1

C 1 8 H 2 0 N +

2 5 0 . 1

5 9 0

2 . 1

9 . 5

M 3 *

[ M + H ] +

7 2 3 . 2

9 0 1

C 3 6 H 4 7 N 6 O 6 S 2 +

7 2 3 . 2

9 9 3

9 . 2

1 6 . 5

+ [ O ] , [ C H 2 ]

a

5 2 5 . 2

5 5 3

C 2 8 H 3 7 N 4 O 4 S +

5 2 5 . 2

5 3 0

2 . 3

1 2 . 5

b

5 0 7 . 2

4 7 8

C 2 8 H 3 5 N 4 O 3 S +

5 0 7 . 2

4 2 4

5 . 3

1 3 . 5

c

4 2 6 . 1

8 6 7

C 2 3 H 2 8 N 3 O 3 S +

4 2 6 . 1

8 4 6

2 . 1

1 1 . 5

d

3 1 1 . 1

7 9 0

C 1 9 H 2 3 N 2 O 2 +

3 1 1 . 1

7 5 4

3 . 6

9 . 5

e

2 9 8 . 1

1 1 7

C 1 3 H 2 0 N 3 O 3 S +

2 9 8 . 1

2 2 0

1 0 . 3

5 . 5

f

2 8 0 . 1

2 0 3

C 1 3 H 1 8 N 3 O 2 S +

2 8 0 . 1

1 1 4

8 . 9

6 . 5

g

2 5 0 . 1

5 1 0

C 1 8 H 2 0 N +

2 5 0 . 1

5 9 0

8 . 0

9 . 5

M 4

[ M + H ] +

7 3 7 . 3

1 0 6

C 3 7 H 4 9 N 6 O 6 S 2 +

7 3 7 . 3

1 5 0

4 . 4

1 6 . 5

+ [ O ]

a

7 1 9 . 2

9 5 7

C 3 7 H 4 7 N 6 O 5 S 2 +

7 1 9 . 3

0 4 4

8 . 7

1 7 . 5

b

5 5 1 . 2

3 5 2

C 2 9 H 3 5 N 4 O 5 S +

5 5 1 . 2

3 2 3

2 . 9

1 4 . 5

c

5 3 3 . 2

2 4 1

C 2 9 H 3 3 N 4 O 4 S +

5 3 3 . 2

2 1 7

2 . 4

1 5 . 5

d

5 0 7 . 2

4 7 8

C 2 8 H 3 5 N 4 O 3 S +

5 0 7 . 2

4 2 4

5 . 3

1 3 . 5

e

4 2 6 . 1

8 5 7

C 2 3

H 2 8

N 3

O 3

S +

4 2 6 . 1

8 4 6

1 . 1

1 1 . 5

S. Jhajra et al .



8/16

T a b l e

3 .

( C o n t i n u e d )

M e t a - b o l i t e

L i n e N o .

M S / T O F d a t a

B e s t p o s s i b l e m o l e c u l a r

f o r m u l a

E x a c t m a s s o f t h e m o s t p r o b a b l e

s t r u c t u r e

E r r o r i n m m u

R D B

M e t a b o l i c c h a n g e

f

3 8 2 . 1

8 4 8

C 2 2 H 2 8 N 3 O S +

3 8 2 . 1

9 4 8

1 0 . 0

1 0 . 5

g

3 1 2 . 1

3 4 9

C 1 4 H 2 2 N 3 O 3 S +

3 1 2 . 1

3 7 6

2 . 7

5 . 5

h

2 9 4 . 1

2 7 5

C 1 4 H 2 0 N 3 O 2 S +

2 9 4 . 1

2 7 1

0 . 4

6 . 5

i

2 8 4 . 1

3 9 4

C 1 3 H 2 2 N 3 O 2 S +

2 8 4 . 1

4 2 7

3 . 3

4 . 5

j

2 5 0 . 1

6 1 0

C 1 8

H 2 0

N +

2 5 0 . 1

5 9 0

2 . 0

9 . 5

M 5

[ M + H ] +

7 3 7 . 3

0 5 7

C 3 7 H 4 9 N 6 O 6 S 2 +

7 3 7 . 3

1 5 0

9 . 2

1 6 . 5

+ [ O ]

a

4 4 2 . 1

7 2 8

C 2 3 H 2 8 N 3 O 4 S +

4 4 2 . 1

7 9 5

6 . 7

1 1 . 5

b

2 9 6 . 1

3 8 7

C 2 8

H 3 5

N 4

O 3

S +

2 9 6 . 1

4 2 7

4 . 0

5 . 5

c

2 6 8 . 1

4 6 3

C 1 3 H 2 2 N 3 O S +

2 6 8 . 1

4 7 8

1 . 5

4 . 5

M 6

[ M + H ] +

7 0 7 . 3

0 4 2

C 3 6 H 4 7 N 6 O 5 S 2 +

7 0 7 . 3

0 4 4

0 . 2

1 6 . 5

[ C H 2 ]

a

5 2 5 . 2

5 5 0

C 2 8

H 3 7

N 4

O 4

S +

5 2 5 . 2

5 3 0

2 . 0

1 2 . 5

b

5 0 7 . 2

4 7 7

C 2 8 H 3 5 N 4 O 3 S +

5 0 7 . 2

4 2 4

5 . 3

1 3 . 5

c

4 8 1 . 2

6 6 2

C 2 7 H 3 7 N 4 O 2 S +

4 8 1 . 2

6 3 2

3 . 0

1 1 . 5

d

4 2 6 . 1

8 6 5

C 2 3 H 2 8 N 3 O 3 S +

4 2 6 . 1

8 4 6

2 . 0

1 1 . 5

e

3 8 2 . 1

9 7 0

C 2 2 H 2 8 N 3 O S +

3 8 2 . 1

9 4 8

2 . 2

1 0 . 5

f

3 6 5 . 1

5 7 1

C 2 2 H 2 5 N 2 O S +

3 6 5 . 1

6 8 2

1 1 . 0

1 1 . 5

g

3 1 1 . 1

6 7 7

C 1 9 H 2 3 N 2 O 2 +

3 1 1 . 1

7 5 4

7 . 7

9 . 5

h

2 8 2 . 1

2 7 1

C 1 3 H 2 0 N 3 O 2 S +

2 8 2 . 1

2 7 1

0 . 0

5 . 5

i

2 6 8 . 1

5 5 9

C 1 8 H 2 2 N O +

2 6 8 . 1

6 9 6

1 3 . 7

8 . 5

j

2 5 0 . 1

6 1 0

C 1 8 H 2 0 N +

2 5 0 . 1

5 9 0

2 . 0

9 . 5

* T h e s e m e t a b o l i t e s w e r e c o n r m e d t o b e s e c o n d g e n e r a t i o n b a s e d o n H R - M S d a t a ; a

l l o t h e r m e t a b o l i t e s w e r e r s t g e n e r a t i o n p r

o d u c t s .

+

i n d i c a t e s a d d i t i o n , w h i l e ( ) i n d i c a t e s l o

s s o f t h e r e s p e c t i v e m o i e t i e s a n d m a s s .

U n p

r e d i c t e d r e p r e s e n t s m o r e t h a n o n e m e t a b o l

i c c h a n g e s .




9/16

However, the exact position of this change could not be proposeddue to low abundance of the product ions, obverting their furtherfragmentation. The fragmentation pattern of the metabolite ispostulated in Fig. 8.

In this case too, CO2 loss occurred during fragment sequences:m/z 551→ 507 and m/z 426→ 382 (shown in the Fig. 8).Evidently, both prescursors of m/z 551 and 426 did not have afree caboxlic group.

Metabolite M5

This metabolite (m/z 737.3073) also had an accurate massdifference of 15.9889 Da (~16 Da) from the parent molecule

(m/z 721.3184). This difference again indicated towards oxidationof the drug. The Q-TOF line spectrum of the metabolite(Supplementary Fig. 1) and its corresponding HRMS (Table 3)and MSn (Table 4) data showed the presence of product ions of m/z 442 and 296, as compared to m/z 426 and 296 in the caseof the drug (Fig. 3). The common product ion of m/z 296(Route 6) revealed that metabolic change did not occur on PartC of the drug. Oppositely, the appearance of a product ion of m/z 442 in the metabolite against characteristic ion of m/z 426(Route 4) observed in the drug clearly indicated that either PartA or B was the site of oxygenation. However, the appearance of a fragment of m/z 250 highlighted that Part B of the drugremained unchanged. So it could be proposed that this mono-oxygenated metabolite appeared upon hydroxylation of partA of the drug molecule. The exact position of change in Part Acould not be delineated, as the key product ions were formed ina very low abundance, obverting their further fragmentation. Thefragmentation pathway of this metabolite is proposed in Fig. 9.

In this case, CO2 loss occurred during fragment sequence: m/z 567→ 523 (shown in the Fig. 9), despite that the precursor wasdevoid of a free caboxlic group.

Metabolite M6

Both HRMS (Table 3) and MSn data (Table 4) were used topropose the structure of metabolite M6. This metabolite(m/z 707.3042) had an accurate mass difference of 14.0142 Dafrom the drug (m/z 721.3184), indicating elemental loss of CH2.The Q-TOF line spectrum for the metabolite is shown in Supple-mentary Fig. 2. The comparison of the fragmentation pattern of the metabolite (Fig. 10) with the drug (Fig. 3) showed that thecharacteristic product ion of m/z 296 (Route 6), representing PartC inthedrug,wasreplaced byionof m/z 282, again with a differenceof 14 Da. This justied the probability of demethylation from Part Cof the drug. Also, the characteristic ion of the drug of m/z 426

(Route 4) and subsequent fragments remained the same in themetabolite, which provided support that there were no changes inParts A and B of the drug during the process of metabolism.

Similar to the parent drug and metabolite M2, neutral CO2 lossoccurred in this metabolite during the fragment sequences: m/z 525→ 481 and m/z 426→ 382 (shown in the Fig. 10), despitethat the precursor ions of m/z 525 and 426 did not have a freecaboxlic group.

Investigation of CO2 elimination during fragmentation of

ritonavir using DFT:

The fragmentation pattern of ritonavir and its metabolites(except in metabolite M1 in which carbamate part was absent

due to metabolic changes) showed neutral loss of CO2 with anaccurate mass difference of ~43.9898 Da in MS2, MS3 or MS4 steps(shown in Figs. 8–12). For example, during MS2 fragmentation of the drug, the parent of m/z 721 formed product ion of m/z 677involving loss of 44 Da. The same mass loss was also observedin a few other transitions in the case of the drug and even themetabolites, viz., m/z 551→ 507, m/z 533→ 489 and m/z 426→ 382. In the absence of a free terminal carboxylate grouppresent in the structures of ions of m/z 721, 551, 533 and 426(Fig. 3), the likely logical explanation for the said loss was eitherrearrangement of the carbamate moiety to a terminalcarboxylate group or the formation of an ion-neutral complex.A critical study of the literature revealed that similar type of neutral loss of CO2 was previously observed by Yu et al.

[12] in

Table 4. MSn fragmentation data for ritonavir metabolites

Metabolite MSn: precursorion(s)*

Product ion(s)*

M1 MS2: 580 562#, 463#, 410, 392, 375, 296,

285, 268, 250#, 197#, 171#

MS3: 410 392, 375#, 347#, 295#, 250

MS3: 296 268 a , 197#, 171#

MS3: 285 268 b , 250#, 233#

MS4: 392 375#, 267#, 250#

MS4: 268a 197#, 171#

MS4: 268b 250#, 233#

M2 MS2: 582 525, 507, 481, 426, 382, 365#, 311,

285, 268, 250#

MS3: 525 507, 481, 311#, 285, 250#

MS3: 426 408#, 382, 365#, 311c, 268, 250#

MS4: 507 446#, 408#, 311#, 250#

MS4: 481 463#, 250#

MS4: 382 365#, 347#, 268#, 250#

MS4: 311c 294#, 267#, 250#

MS4: 285 268#, 250#

MS4

: 268 250#

M3 MS2: 723 525, 507#, 426, 311, 298, 280#, 250#

MS3: 525 507#

MS3: 426 408#, 382#, 365#, 311, 250#

MS3: 298 280#

MS4: 311 250#

M4 MS2: 737 719#, 551, 533, 507#, 426, 382#,

311#, 284, 250#

MS3: 551 533, 507#, 489#, 408#, 311#, 250#

MS3: 426 408#, 382#, 365#, 268#, 250#

MS3: 312 284#, 213#

MS4: 533 507#

M5 MS2: 737 719#, 567, 549#, 523#, 442, 311#,

296, 268

MS2: 567 549#,523#, 505#

MS3: 523 392#, 311#

MS3: 442 424, 311#, 250#

MS3: 296 268, 197#, 171#

MS4: 268 197#, 171#

M6 MS2: 707 525, 507#, 481#, 426, 282

MS3: 525 507#,481#

MS3: 426 408#, 382#, 311#, 268#, 250#

MS3: 282 –

* All values in m/z ; #Ions could not be captured for further MSn

studies; superscripts a, b: Ions of same mass that on subsequentMSn analysis yielded different fragments.

S. Jhajra et al .



10/16

carbamate compounds. Also, several reports were found, whichmentioned of unusual neutral losses like SO, SO2, H2SO2, CO, H2O,

NH3 , etc . from the central part of protonated or deprotonatedions.[13–22] Many of these investigationsalso proposed mechanismsof occurrence of such unusual losses. In most studies involving lossof SO, SO2, H2SO2, CO and H2O, the proposition was a gaseousphase rearrangement, which was hypothesized to be energeticallyfavoured.[13–21] The elimination of NH3 was justied through anion–neutral complex formation.[22]

Therefore, the mechanism during the mass fragmentationprocess in our case was explored considering both, therearrangement and the ion–neutral complex formation. Thiswas done by quantum chemical analysis through DFT, an in silicotool that had been employed by many researchers for the samepurpose, but in different situations.[23,24] The basic contentionemployed was that the fragmentation pattern of the parent ion

was inuenced by factors like 3D conguration, stability andinternal energy during its dissociation in the collision cell. [25–28]

Also, DFT had been reported to be very well applicable togas-phase chemistry that prevailed in the MS system and couldbe used to calculate electron density over a neutral molecule,and redistribution of the same, and even its inuence on bondstrengths once the protonation has occurred.[23,29] Further, it ishelpful in the optimization of structural geometries of moleculesbased on energy minimization calculations and even assists inthe prediction of the preferable sites of protonation amongstseveral possibilities (relative proton af nities), including thedetermination of energy redistribution and bond labilities.[23,29]

For DFT calculations, a structural part common to ritonavir andmass fragments of m/z 551, 533 and 426 was selected as aprecursor ion (PI1+) model system. Figure 11 shows possiblepathways A and B for the CO2 loss involving rearrangement

Figure 5. Proposed mass fragmentation pattern of metabolite M1 of ritonavir in ESI positive ionization mode.




11/16

and ion–neutral complex, respectively. The potential energy of PI1+ was arbitrarily considered as zero for the calculation of relative energies of the intermediates (I1+–I4+), transition states(TS1–TS4, Fig. 12) and the product ion (Pr+). The correspondingpotential energy diagrams of both the pathways A and B areshown in Fig. 13.

As the parent had multiple potential sites (O3, O4, N2, N48) forprotonation, the proton af nity values at these centres wereestimated. The results (see Supplementary Table 2) indicated thatN48 of thiazole ring was the most preferred site for protonation,in comparison to others. Hence, N48+ ion (PI1+) was chosen as aprecursor to develop the reaction pathway. Incidentally, PI1+

could exist in iminol tautomeric state represented by I1+, forwhich further two conformations (extended and closed) werepossible, as shown in pathway A in Fig. 11. Evidently, the closedconformation was stable by 9.11 kcal/mol, and its closed geome-try assisted in the rearrangement, forming TS1 (3D structure inFig. 12) through simultaneous bond breaking (C45―O4) and

bond formation (C45―

N2), with a transition state barrier of 54.68kcal/mol. TS1 further formed intermediate (I2+) with apotential energy of 6.37 kcal/mol with reference to PI1+. Thisintermediate converted to product ion (Pr+) with an activationenergy of 35.17 kcal/mol through TS2 (3D structure in Fig. 12)by losing CO2 through cleavage of C1―N2 bond (d C1―N2=1.56Å)and simultaneous shift of the proton from O4 to N2. As shown inFig. 13, this step represented the lowest energy point in theentire reaction pathway A, with potential energy value of 5.31kcal/mol, highlighting that CO2 loss was a thermodynamicallydriven process.

The ion–neutral complex is proposed to form upon transfer of proton from ring nitrogen in CID to O4 via a through spaceinteraction in PI1+ or perhaps pre-existence of charge on O4, as

part of the possible multiple protonation sites in the parent, P(Pathway B). The resultant ion is shown as PI2+ in Fig. 11, whichevidently was less stable as compared to PI1+ by 34.73 kcal/mol(see Table 2, supporting information). In PI2+, the C45―O4 bond(bond dissociation energy (BDE) of 8.71 kcal/mol) underwentcleavage to form I3+, an ion–neutral complex between thiazolemethylium cation and carbonic acid. This complex after surpass-ing energy barrier of 15.81 kcal/mol converted to intermediateI4+ by the formation of C45―N2 bond due to the attack of N2

centre on the electron decient carbon (C45) of methyliumcation as shown in TS3 (3D structure in Fig. 12). The intermedi-ate I4+ is characterized by quaternary nitrogen centre carrying aCOOH moiety. The elimination of CO2 from this intermediatehappened through proton migration, justied through attrac-tion of the latter (during the loss of CO2) towards the π-cloudof the aromatic thaizole ring. The result was the formation of another transition state, TS4. The transfer of carbamate COOHproton with concomitant cleavage of the N-CO2 bond

eventually led to the formation of product ion (Pr+

). This pathwayB, thus, is also thermodynamically driven.

A critical comparison of pathways A and B revealed that due toless stability of PI2+ than PI1+ by 34.73 kcal/mol, the pathway Awas favoured initially. However, the consideration of overallenergy barrier highlighted that during pathway A, there wassubsequent hurdle of ~73 kcal/mol, while it was a lower valueof ~41 kcal/mol in pathway B. This meant that pathway B wasthe more likely mechanism involved. Other reasons dictatingthe preference for pathway B included the possibility of delocali-zation of cation on the methyl thiazole ion, thus improving itsstability, and subsequent π–π interaction between thiazolemethylium ion and the phenyl ring of Part B of drug (Fig. 1) inthe neutral moiety after loss of CO2.

Figure 6. Proposed mass fragmentation pattern of metabolite M2.

S. Jhajra et al .



12/16

Figure 7. Proposed mass fragmentation pattern of metabolite M3 in ESI positive ionization mode.





13/16



S. Jhajra et al .



14/16

Investigational analysis of differential fragmentation behaviour

of ritonavir and its metabolites by using DFT

A critical appraisal of the fragmentation pathways of ritonavir(Fig. 3) and its metabolites M1–M6 (Figs. 5 to 10) showed a differ-ential behaviour. Ritonavir and its metabolites, M1, M4 and M5,which had intact tertiary N31, underwent cleavage of C30―N31bond, as exemplied through conversion of parent to production of m/z 551 in the case of the drug (Route 2). On the otherhand, metabolites M2, M3 and M6, in which N31 was convertedto secondary form as a result of demethylation or deacylation,followed cleavage of N29―C30 bond. In order to understand thisdifferential behaviour during fragmentation, quantum chemical

calculations were again performed by using DFT. The modelstructures taken for DFT calculations are shown in Figs. 14a andb. The results are given in Table 5. The minor difference in relativeenergy values under gas phase condition suggested that proton-ated ions N29+ and N31+ were isoenergetic to each other. In thecase of implicit solvent analysis, which was done by taking ACNas a solvent (major component in the mobile phase), the relativestability of the two protonated ions differed by 9.68 kcal/mol,with N31+ being considerably more stable. It highlighted thatthe fragmentation of C30―N31 bond was energetically preferredfor ritonavir and its metabolites M1, M4 and M5, in line with theexperimental observation. The same was also supported by cal-culated proton af nity values in gas and solvent phase conditions

Figure 11. Plausible reaction pathways for the loss of CO2 through the rearrangement (pathway A) and ion –neutral mechanism (pathway B). The 3Doptimized geometries for whole reaction pathway are given in Supplementary Figs. 4 –5. All values are in kcal/mol.

TS4TS3TS1 TS2

Figure 12. 3D optimized geometries of transition states involved in reaction pathways A and B. Dashed lines represents bond distances in Å unit.

-20

-10

0

10

20

30

40

50

60

70

80

(Pathway B) (Pathway A)

E n e r g y ( k c a l / m o l )

Figure 13. Potential energy surface for the reaction pathways A and B involving the loss of CO2 from the precursor ion. Energy of I1+ in closed con-

formation was taken under consideration. The energies are in kcal/mol relative to the precursor ion (PI1 +).




15/16

(Table 5). The respective values were 220.83kcal/mol and242.75 kcal/mol for N29+, and 221.45 kcal/mol and 252.47 kcal/ mol for N31+. Hence, this data also supported better stability of the latter protonated ion. On the contrary, in the case of M2, M3and M6 metabolites, N29+ ion was signicantly stable even underthe gas phase condition by 10.68 kcal/mol, and it also maintainedits thermodynamic stability in the investigated solvent condition(Table 5). The predominance of N29+ ion over N31+ was alsosupported by the higher proton af nity value for the attack of proton on N29 centre, which supported the dissociation of N29―C30 bond in demethylated metabolites of ritonavir.

In the literature, a study of the effect of N -substitution on N―Cbond strength of amide group by using DFT and a comparisonwith experimentally calculated BDE was reported by Marochkinet al.[30] They found that electron-donating substituents like ―C(O)―, ―NHC(O)―, ―O― and ―N3 on carbonyl of the amidebond had stabilizing effect and resulted in increase in BDE.However, the substitution of groups like methyl and phenyl onthe amide nitrogen resulted in decrease in BDE by weakeningof the N―C bond. Substituted methyl and phenyl decreasedBDE by 10–90 kJ/mol on moving from primary to tertiary amides.

Therefore, the corroboration of the above-discussed differentialbehaviour was done through BDE studies, considering that disso-ciation of C30―N31 or N29―C30 bond was also governed by thestability of resultant fragments after bond cleavage. In the case of ritonavir and its metabolites, M1, M4 and M5, the fragments thatwere formed owing to the dissociation of C30 ―N31 bond werecharacterized by charge stabilization due to delocalization onN29, C30 and O32 atomic centres. The difference in the chargedensity between N29 and N31 centres was accordingly foundto be 0.206 e. However, in demethylated analogues, the differ-ence in the charge density (N29 and N31) was greatly reduced to0.074 e, making both the sites equally electron dense. Thus,proton af nity values at N29 and N31 (Table 5) were decisive of preferable protonation site, as discussed above.

Summary

The present study provides extensive biotransformation-related infor-mation on ritonavir, along with extensive mass fragmentationpathways of the drug and its all six CYP3A4 generated metabolites.The metabolitesformed were deacylated ritonavir (M1), N -dealkylatedritonavir (M2), N -demethylated C -hydroxylated ritonavir (M3),C -hydroxylated ritonavir (M4 and M5) and N -demethylated ritonavir

(M6) of which two were new. The unusual CO2 loss during mass frag-mentation of ritonavir was analyzed for possibility of rearrangementas well as ion-neutral complex mechanism through DFT. Althoughthe initial step for protonation appeared to favour pathway A(rearrangement mechanism), favourable activation barriers found onthe reaction pathway of ion–neutral mechanism suggest that thispathway is credible for CO2 loss, as justied by DFT. In addition, thedifference in fragmentation pathways of ritonavir and its metabolites(M1 to M6) could be ascribed to difference in the proton af nityvalues on N29 and N31 centres, again justied by DFT modelling.

Acknowledgements

Authors want to acknowledge Bristol Myers Squibb, Bangalore,India for providing fellowship and nancial assistance to one of the authors (Shalu Jhajra). Authors also acknowledge NinadVarkhede (Biocon-Bristol Myers Squibb Research Center (BBRC,Bangalore)), Ravi Shah (BBRC, Bangalore) and reviewers of thismanuscript for their valuable suggestions.

References

[1] B. Prasad, S. Singh. Identication of rat urinary metabolites of rifabutinusing LC–MSn and LC–HR-MS. Eur. J. Pharm. Sci. 2010, 41, 173.

[2] B. Prasad, A. Garg, H. Takwani, S. Singh. Metabolite identication byliquid chromatography-mass spectrometry. TrAC Trends Anal. Chem.2011, 30, 360.

[3] B. Prasad, S. Singh. In vitro and in vivo investigation of metabolic fate

of rifampicin using an optimized sample preparation approach andmodern tools of liquid chromatography–mass spectrometry.

J. Pharm. Biomed. Anal. 2009, 50, 475.[4] H.-l. Lin, J. D’Agostino, C. Kenaan, D. Calinski, P. F. Hollenberg. The ef-

fect of ritonavir on human CYP2B6 catalytic activity: Heme modica-tion contributes to the mechanism-based inactivation of CYP2B6and CYP3A4 by ritonavir. Drug Metab. Dispos. 2013, 41, 1813.

[5] R. G. Parr, W. Yang. Density-functional theory, Oxford universitypress: New York, 1989.

[6] A. D. Becke. Density-functional thermochemistry. III. The role of exactexchange. J. Chem. Phys. 1993, 98, 5648.

[7] C. Lee, W. Yang, R. G. Parr. Development of the Colle-Salvetticorrelation-energy formula into a functional of the electron-density. Phys. Rev. B. 1988, 37 , 785.

[8] M. J. Frisch, G. W. Trucks, H. B. Schlegel, G. E. Scuseria, M. A. Robb, J. R.Cheeseman, G. Scalmani, V. Barone, B. Mennucci, G. A. Petersson, H.

Nakatsuji, M. Caricato, X. Li, H. P. Hratchian, A. F. Izmaylov, J. Bloino,G. Zheng, J. L. Sonnenberg, M. Hada, M. Ehara, K. Toyota, R. Fukuda,J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda, O. Kitao, H. Nakai,T. Vreven, J. A. Montgomery Jr., J. E. Peralta, F. Ogliaro, M. Bearpark,J. J. Heyd, E. Brothers, K. N. Kudin, V. N. Staroverov, R. Kobayashi,J. Normand, K. Raghavachari, A. Rendell, J. C. Burant, S. S. Iyengar,J. Tomasi, M. Cossi, N. Rega, J. M. Millam, M. Klene, J. E. Knox,J. B. Cross, V. Bakken, C. Adamo, J. Jaramillo, R. Gomperts, R. E. Stratmann,O. Yazyev, A. J. Austin, R. Cammi, C. Pomelli, J. W. Ochterski, R. L. Martin,K. Morokuma, V. G. Zakrzewski, G. A. Voth, P. Salvador, J. J. Dannenberg,S. Dapprich, A. D. Daniels, Ö. Farkas, J. B. Foresman, J. V. Ortiz,J. Cioslowski, D. J. Fox. Gaussian 09: EM64L-G09RevB.01, Gaussian Inc.:Wallingford CT, 2010.

[9] A. P. Scott, L. Radom. Harmonic vibrational frequencies: An evalua-tion of Hartree-Fock, Møller-Plesset, quadratic conguration interac-tion, density functional theory, and semiempirical scale factors.

J. Phys. Chem. 1996, 100, 16502.

Figure 14. Model structures used for analysis of differential fragmenta-tion behaviour of ritonavir and its metabolites by using DFT.

Table 5. Relative energy (RE) and proton af nity (PA) values (kcal/mol)of different ions calculated using B3LYP/6-31+ g(d) level of theory

RE PA

Gasphase

Solventphase*

Gasphase

Solventphase*

Fig. 14a-N29+ 0.57 9.68 220.83 242.75

Fig. 14a-N31+ 0.00 0.00 221.45 252.47

Fig. 14b-N29+ 0.00 0.00 220.53 251.04

Fig. 14b-N31+

10.68 7.83 209.68 243.27

* Acetonitrile was chosen as solvent as it was the majorcomponent of the mobile phase.

S. Jhajra et al .



16/16

[10] A. E. Reed, L. A. Curtiss, F. Weinhold. Intermolecular interactionsfrom a natural bond orbital, donor-acceptor viewpoint. Chem.Rev. 1988, 88 , 899.

[11] A. E. Reed, R. B. Weinstock, F. Weinhold. Natural population analysis. J. Chem. Phys. 1985, 83, 735.

[12] D. Yu, Q. Feng, Z. Yu. An unusual rearrangement process of neutralloss of CO2 in carbamates by electrospray ionization-multistagemass spectrometry. J. Chin. Mass Spectrom. Soc. 2005, 26, 6.

[13] M. Sun, W. Dai, D. Q. Liu. Fragmentation of aromatic sulfonamides in

electrospray ionization mass spectrometry: Elimination of SO2 viarearrangement. J. Mass Spectrom. 2008, 43, 383.[14] K. Klagkou, F. Pullen, M. Harrison, A. Organ, A. Firth, G. J. Langley.

Fragmentation pathways of sulphonamides under electrospray tan-dem mass spectrometric conditions. Rapid Commun. Mass Spectrom.2003, 17 , 2373.

[15] N. Hu, P. Liu, K. Jiang, Y. Zhou, Y. Pan. Mechanism study of SO2 elim-ination from sulfonamides by negative electrospray ionization massspectrometry. Rapid Commun. Mass Spectrom. 2008, 22, 2715.

[16] S. Crotti, L. Stella, I. Munari, F. Massaccesi, L. Cotarca, M. Forcato, P.Traldi. Claisen rearrangement induced by low-energy collision of ESI-generated, protonated benzyloxy indoles. J. Mass Spectrom.2007, 42, 1562.

[17] F. Wang. Collision-induced gas-phase Smiles rearrangement inphenoxy-N -phenylacetamide derivatives. Rapid Commun. MassSpectrom. 2006, 20, 1820.

[18] Y. Zhou, Y. Pan, X. Cao, J. Wu,K. Jiang. Gas-phase smiles rearrangement

reactions of deprotonated 2-(4, 6-dimethoxypyrimidin-2-ylsulfanyl)-N -phenylbenzamide and its derivatives in electrospray ionization massspectrometry. J. Am. Soc. Mass Spectrom. 2007, 18, 1813.

[19] X. Chen, J. Xing, D. Zhong. Rearrangement process occurring inthe fragmentation of adefovir derivatives. J. Mass Spectrom.2004, 39 , 145.

[20] Z. X. Zhao, H. Y. Wang, C. Xu, Y. L. Guo. Gas-phase synthesis of hydrodiphenylcyclopropenylium via nonclassical Favorskii rearrangementfrom alkali-cationizedα, α′ -dibromodibenzyl ketone. Rapid Commun. MassSpectrom. 2010, 24, 2665.

[21] Y. Chen, Y. Yin, X. Sun, X. Liu, H. Wang, Y. Zhao. A novelrearrangement in electrospray ionization multistage tandem massspectrometry of amino acid ester cyclohexyl phosphoramidates of AZT. J. Mass Spectrom. 2005, 40, 636.

[22] J. Bialecki, J. Ruzicka, A. B. Attygalle. An unprecedentedrearrangement in collision-induced mass spectrometric fragmenta-tion of protonated benzylamines. J. Mass Spectrom. 2006, 41, 1195.

[23] A. Alex, S. Harvey, T. Parsons, F. S. Pullen, P. Wright, J. A. Riley.Can density functional theory (DFT) be used as an aid to a

deeper understanding of tandem mass spectrometricfragmentation pathways? Rapid Commun. Mass Spectrom.2009, 23, 2619.

[24] Z. Xiang. Mechanism of SO2 elimination from the aromaticsulfonamide anions: A theoretical study. Comput. Theor. Chem.2012, 991, 74.

[25] A. K. Shukla, J. H. Futrell. Tandem mass spectrometry: Dissociation of ions by collisional activation. J. Mass Spectrom. 2000, 35, 1069.

[26] E. De Hoffmann. Tandem mass spectrometry: A primer. J. MassSpectrom. 1996, 31, 129.

[27] V. Gabelica, E.D. Pauw. Internal energy and fragmentation of ions produced in electrospray sources. Mass Spectrom. Rev.2005, 24 , 566.

[28] C. Collette, L. Drahos, E. D. Pauw, K. Vékey. Comparison of the inter-nal energy distributions of ions produced by different electrospraysources. Rapid Commun. Mass Spectrom. 1998, 12, 1673.

[29] M. Alcamí, O. Mo, M. Yáñez. Computational chemistry: A useful

(sometimes mandatory) tool in mass spectrometry studies. MassSpectrom. Rev. 2001, 20, 195.

[30] I. I. Marochkin, O. V. Dorofeeva. Amide bond dissociation enthalpies:Effect of substitution on NAC bond strength. Comput. Theor. Chem.2012, 991, 182.

Supporting information

Additional supporting information may be found in the onlineversion of this article at the publisher’s web site.


Date post:	01-Jun-2018
Category:	Documents
Upload:	prasanth-bitla
View:	236 times
Download:	0 times

DFT for ritonavir.pdf

Documents