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Journal of Chromatography B, 879 (2011) 1–7

Contents lists available at ScienceDirect

Journal of Chromatography B

journa l homepage: www.e lsev ier .com/ locate /chromb

vidence of the formation of direct covalent adducts of primaquine,-tert-butylprimaquine (NP-96) and monohydroxy metabolite of NP-96 withlutathione and N-acetylcysteine

mit Garga, Bhagwat Prasada, Hardik Takwania, Meenakshi Jainb, Rahul Jainb, Saranjit Singha,∗

Department of Pharmaceutical Analysis, National Institute of Pharmaceutical Education and Research (NIPER), Sector 67, S.A.S. Nagar 160062, Punjab, IndiaDepartment of Medicinal Chemistry, National Institute of Pharmaceutical Education and Research (NIPER), Sector 67, S.A.S. Nagar 160062, Punjab, India

r t i c l e i n f o

rticle history:eceived 14 July 2010ccepted 27 October 2010vailable online 4 November 2010

eywords:rimaquineP-96

a b s t r a c t

2-tert-Butylprimaquine (NP-96) is a novel quinoline anti-malarial compound with superior therapeuticprofile than primaquine (PQ). Moreover, it is the first 8-aminoquinoline that is established to be devoid ofmethemoglobin toxicity. The purpose of the present study was to investigate covalent adduct formationtendency of PQ, NP-96 and their phase I metabolites with glutathione (GSH) and N-acetylcysteine (NAc).For the same, the two compounds were incubated in human and rat liver microsomes in the presenceof trapping agents and NADPH. In a control set, NADPH was excluded, while a blank was also studiedthat was devoid of both NADPH and microsomes. The components in the reaction mixtures were initially

lutathione-Acetylcysteineeactive metaboliteC–MS

separated on a C-18 column (250 mm × 4.6 mm, 5 �m) using a mobile phase composed of acetonitrileand 10 mM ammonium acetate in a gradient mode. The samples were then subjected to LC–MSn andLC–HR-MS analyses, and data were collected in full scan MS, data dependent MS/MS, targeted MS/MS,neutral loss scan (NLS) and accurate mass (MS/TOF) modes. In a significant finding, both PQ and NP-96themselves showed potential to bind covalently with GSH and NAc, as adducts were observed even inthe control and blank incubations. Intense peaks corresponding to covalent adduct of mono-hydroxy

GSH

metabolite of NP-96 with

. Introduction

Most widely employed drugs for the treatment of malaria arehloroquine, primaquine (PQ) and artemisinin. However, all thesere associated with one problem or the other. The use of chloro-uine has been questioned owing to emergence of resistance anddverse effects [1]. PQ is employed for prophylaxis, but showsigh incidence of toxicity, particularly in individuals with glucose--phosphate dehydrogenase (G6PD) deficiency [2]. Artemisinin

s normally prescribed in combination therapy with other anti-alarials due to its shorter duration of action, but its use is also

estricted due to the risk of development of bacterial resistance3]. Thus considering the hurdles in therapy of malaria with estab-ished drugs, there is an urgent need for development of molecules

ith improved therapeutic activity and better safety to combat the

readful onslaught of the parasite.

2-tert-Butylprimaquine (NP-96, Fig. 1) is a new experimentalnti-malarial candidate, which is reported to be more potent andafer than PQ. It showed promising activity in vitro and in vivo, and

∗ Corresponding author. Tel.: +91 172 2292031; fax: +91 172 2214692.E-mail address: ssingh@niper.ac.in (S. Singh).

570-0232/$ – see front matter © 2010 Elsevier B.V. All rights reserved.oi:10.1016/j.jchromb.2010.10.029

and NAc were also detected in NADPH supplemented reaction solution.© 2010 Elsevier B.V. All rights reserved.

more importantly it was devoid of methemoglobin (MetHB) toxic-ity [4]. Subsequent studies showed that it had schizonticidal actionin vivo [5]. In continued investigations on this new medicinal entity(NME), the reactive metabolite formation potential was explored inthe present study. The same was considered important keeping intoview reports on the formation of direct covalent adducts of substi-tuted quinolines with nucleophiles [6,7]. The study utilized variousmodes of LC–MS to detect and identify the adducts [8].

2. Materials and methods

2.1. Materials

NP-96 was synthesised and supplied by the Department ofMedicinal Chemistry, NIPER, S.A.S Nagar, India. Rat liver micro-somes (RLM) were purchased from BD Biosciences (San Jose, USA).Human liver microsomes (HLM) were supplied gratis by CellzDi-rect (Durham, USA). Nicotinamide adenine dinucleotide phosphate

reduced tetrasodium salt (NADPH), di potassium hydrogen phos-phate and potassium phosphate monobasic were purchasedfrom Himedia Laboratories (Mumbai, India). Primaquine, glu-tathione (GSH) and N-acetylcysteine (NAc) and sodium formatewere procured from Sigma–Aldrich Chemicals (Bangalore, India).

2 A. Garg et al. / J. Chromatog

N

H3CO

HNNH3

-17 Da

-85 Da

17

5.0

86

3

24

3.1

48

7

26

0.1

75

3

+MS, 9.1-9.8min

0.00

0.25

0.50

0.75

1.00

5x10

Intens.

140 160 180 200 220 240 260 m/z

(A) (A)(A)A

N

H3CO

HN NH3

-17 Da

-85 Da

C(CH3)3

23

1.1

48

3

29

9.2

10

6

31

6.2

37

8

+MS, 5.0-5.4min

0

1

2

3

4

5

5x10

Intens.

240 260 280 300 m/z

(B) (B)(B)B

24

7.1

42

8

28

4.3

27

8

31

5.2

05

7

33

2.2

33

3

+MS, 20.9min

2000

4000

6000

Intens.

N

H3CO

HNH2C NH3

C(CH3)3

-17 Da

-85 Da

OH(C)

N

H3CO

HNH2C NH3

C(CH3)3

-17 Da

-85 Da

OH(C)(C)

N

H3CO

HN NH3

C(CH3)3

-17 Da

-85 Da

OH

N

H3CO

HNH2C NH3

C(CH3)3

-17 Da

-85 Da

OHC

Fa

APpoA

TH

0

240 260 280 300 320 m/z

ig. 1. HR-MS spectra, structures and sites of fragmentation of PQ (A), NP-96 (B)nd monohydroxy-NP-96 (C).

cetaminophen was obtained as a gratis sample from Arbroharmaceuticals (Delhi, India). HPLC grade acetonitrile (ACN) wasrocured from J.T. Baker (Phillipsburg, NJ, USA). Pure water wasbtained from a water purification unit (Elga Ltd., Bucks, England).ll other chemicals used were of analytical-reagent grade.

able 1PLC gradient programmes and MSn and MS/TOF parameters used for the test molecules

PQ NP-96

Time (min) Buffer (%) Time (min)

0 → 3 97 0 → 23 → 10 97 → 75 2 → 1310 → 16 75 → 60 13 → 2316 → 23 60 → 20 23 → 3023 → 26 20 → 97 30 → 3226 → 40 97 32 → 45

MSn parameters PQ

Sheath gas (arb) 30Auxillary gas (arb) 10Spray voltage (kV) 5Capillary temperature (◦C) 300Capillary voltage (V) 48Tube lens voltage (V) 40

MS/TOF parameters PQ-GSH

Low mass (m/z) 100Collision energy (eV/Z) 10Collision RF (Vpp) 300Transfer time (�s) 92

r. B 879 (2011) 1–7

2.2. Incubation conditions and sample preparation

Liver microsomes (1 mg/ml), test compounds (10 �M), MgCl2(3.3 mM), NADPH (1.3 mM) and GSH or NAc (5 mM) were incu-bated at 37 ± 1 ◦C for 60 min. The control was a mixture devoidof NADPH, while, in blank both NADPH and microsomes wereexcluded. Also, acetaminophen was used as a positive control forall the microsomal incubations. After the presumed time, aliquotswere withdrawn and subjected to sample preparation before anal-ysis by LC–MS. Sample preparation step mainly involved proteinprecipitation using chilled ACN, followed by centrifugation at9000 × g for 10 min (Eppendorf, Hamburg, Germany). The sam-ples were further purified by solid phase extraction (SPE) usinghydrophilic–lipophilic balance (HLB) cartridges (Waters, Milford,USA). The procedure involved cartridge conditioning with 1 mlmethanol, followed by 2 ml water, loading of samples (1 ml), wash-ing with 1 ml water and elution of the analytes with 0.5 ml ACN.Finally, aliquots from SPE were pooled and evaporated to drynessunder a stream of nitrogen at 40 ◦C in a TurboVap (Caliper LifeSciences, Hopkinton, USA) and stored at −60 ◦C until analyses.

2.3. LC method development

During LC method development, the main consideration wasgood resolution between test molecules, their metabolites andadducts with GSH/NAc. To achieve the same, mobile phase wasoptimized in a manner that the test compound resolved at ∼15 minon a C-18 column and other polar components eluted before it,with good resolution among themselves. For LC–MS studies, themethods were suitably modified by using a volatile buffer in themobile phase. The desired separation was achieved on a ZorbaxC-18 column (250 mm × 4.6 mm, 5 �m) from Agilent Technolo-gies (Wilmington, DE, USA), employing a gradient mode and amobile phase comprising of ACN and 10 mM ammonium acetate(pH 5.0). Details of the gradient methods are given in Table 1. In allthe studies, the flow rate and column temperature were fixed as0.35 ml/min and 25 ◦C, respectively.

2.4. LC–MS studies

LC–MSn studies were performed on LTQ-XLTM linear ion-trapequipment (Thermo Electron Corporation, San Jose, USA) controlledwith Xcalibur (version 2.0) software coupled to AccelaTM HPLC

(PQ and NP-96) and positive control (acetaminophen).

Acetaminophen

Buffer (%) Time (min) Buffer (%)

95 0 → 4 9795 → 55 4 → 14 97 → 2055 → 5 14 → 18 205 18 → 20 20 → 975 → 95 20 → 30 9795

NP-96

3012

5275

3165

PQ-NAc NP-96-GSH NP-96-NAc

80 100 8010 10 10

300 400 30092 100 100

A. Garg et al. / J. Chromatogr. B 879 (2011) 1–7 3

Simultaneous data acquisition in full scan and data-dependent

modes

Preliminary detection of adducts based on molecular ion peaks

of M+305 (GSH) or M+161(NAc), NLS and MS/MS spectra

Targeted MS/MS scan in positive mode

Removal of false positives based on NLS (129 Da) for GSH

and MS/MS spectra matching

Targeted MS/MS scan in negative mode

Further confirmation based on characteristic product ion of

m/z 272 in GSH and [M+31] in NAc

acqu

Injection 1

Injection 2

Injection 3

H and

sptsiMc(DbCm

TH

Data Injection 4

Fig. 2. Strategy employed for identification of GS

ystem. Mass ionization parameters were optimized to get bestossible fragmentation with acceptable sensitivity. Various detec-ion modes, like selective reaction monitoring (SRM), neutral losscan (NLS) and targeted MS/MS were utilised for the detection anddentification of adducts. High resolution mass spectrometry (HR-

S) studies were carried out using a LC-MS/TOF instrument, whichomprised of 1100 series HPLC system from Agilent Technologies

Waldbronn, Germany) and MicrOTOF-Q spectrometer from Brukeraltonics (Bremen, Germany). The two were operated using com-ined software comprising of Hystar (version 3.1) and MicrOTOFontrol (version 2.0). The calibration solution used for accurateass studies was 5 mM sodium formate. All masses were corrected

able 2R-MS data for PQ, NP-96, GSH and NAc.

Fragment Experimental mass Best possible molecular f

PQM+H+ 260.1753 C15H22N3O+

(M+H+)-NH3 243.1487 C15H19N2O+

(M+H+)-C5H11N 175.0863 C10H11N2O+

NP-96M+H+ 316.2377 C19H30N3O+

(M+H+)-NH3 299.2106 C19H27N2O+

(M+H+)-C5H11N 231.1483 C14H19N2O+

Monohydroxy-NP-96M+H+ 332.2333 C19H31N3O2

+

(M+H+)-NH3 315.2057 C19H28N2O2+

(M+H+)-C5H11N 247.1428 C14H20N2O2+

GSHM+H+ 308.0892 C10H18N3O6S+

(M+H+)-H2O 290.0768 C10H16N3O5S+

(M+H+)-C2H5NO2 233.0580 C8H13N2O4S+

(M+H+)-C5H7NO3 179.0483 C5H11N2O3S+

NAc(M+H+) 164.0358 C5H10NO3S+

(M+H+)-H 146.0258 C5H8NO2S+

(M+H+)-C2H3O 122.0278 C3H8NO2S+

isition in HR-MS mode

NAc adducts. M = m/z value of parent compound.

by internal reference ions having m/z values of 90.9766, 158.9641,226.9515, 362.9263, 430.9138, 498.9012, 566.8886, 634.8760 and702.8635. Both the above systems were used to study mass frag-mentation behavior of the precursors, i.e., PQ, NP-96, GSH and NAc,and also for the analyses of experimental samples. The optimizedmass parameters are given in Table 1.

2.5. Detection and identification of covalent adducts

For the detection and confirmation of GSH/NAc adduct(s), theprotocol given in Fig. 2 was followed [9–14]. Each sample was sub-jected to data acquisition in full scan and data-dependent positive

ormula Exact mass of most possible structure Error in ppm

260.1757 1.537243.1487 0.000175.0866 1.713

316.2383 1.581299.2118 4.010231.1492 3.893

332.2333 0.000315.2067 3.172247.1441 5.260

308.0911 6.167290.0805 12.755233.0591 4.719179.0485 1.117

164.0376 10.973146.0270 8.217122.0270 6.555

4 A. Garg et al. / J. Chromatogr. B 879 (2011) 1–7

1 2

12 14 16 18 20 22 24 26 28 30 32

0

10

20

30

40

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100R

elat

ive

Abundan

ce

Time (min)

A

14 16 18 20 22 24 26 28

0

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40

60

80

100

1 2

Rel

ativ

e A

bundan

ce

Time (min)

B

200 300 400 500

m/z

0

50

100

272

254

545

179

563

434

210

290

466

521

Rel

ativ

e A

bundan

ce A2

200 300 400 500 600

m/z

0

20

40

60

80

100

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254

601

490

210

346

575

444

Rel

ativ

e A

bundan

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404

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m/z

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48

0

52

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56

6

36

8

22

6

46

4

Re

lative

Abun

dance

A1

56

5

200 300 400 500 600

m/z

0

50

100

49

2

62

1

40

7

31

6

28

8

43

0

57

6

34

6

36

5

23

1

52

9

55

7

26

7

Re

lati

ve A

bu

nd

an

ce

B1

6044752

99

460

536

F -GSHi 6-GSH

MmhvtaoeaNi

ig. 3. Neutral loss (129 Da) chromatograms of PQ-GSH (m/z 565) (A) and NP-96onization modes for PQ-GSH (m/z 565 and 563, respectively) (A1 and A2) and NP-9

S/MS, targeted MS/MS (both ESI positive and negative ionizationodes), and HR-MS modes. Full scan and data-dependent MS/MS

elped in predicting the number of possible adducts and their m/zalues. The data acquired in positive ESI targeted MS/MS mode forhe predicted values helped in identification of molecular ions ofdducts with GSH and determining their number through NLS scan

f 129 Da. The negative targeted MS/MS data reconfirmed the pres-nce and number of adducts by the appearance of ions of m/z 272nd 254 for GSH adducts [15], and typical fragments in case ofAc adducts. Both studies thus assisted in eliminating false pos-

tives [16]. Finally, identification of each adduct was assisted by its

NH2

CHO

O

OHN

O

S

NH

CH

O

c

d

f

H3COe

Adducts R M M-a M-b M-c M-d M-(d+a)

PQ-GSH H 565 548 480 436 419 404

NP-96-GSH C(CH3)3 621 604 536 492 475 460

Key: M denotes (M+H)+, a-f represent leaving fragments

Fig. 4. Structure of adducts of PQ and NP-96 with GSH a

(m/z 621) (B) along with representative MS/MS spectra in positive and negative(m/z 621 and 619, respectively) (B1 and B2).

HR-MS data, which were used to calculate their elemental compo-sitions.

3. Results and discussion

3.1. Fragmentation patterns of test compounds and nucleophiles

The HR-MS spectra of PQ and NP-96 are shown in Fig. 1, alongwith their fragmentation profiles. Both compounds underwentfacile two-step fragmentation in ESI positive mode, involving com-mon losses of 17 Da (NH3) and 85 Da (aliphatic side chain). In case

N

OH

NH3+

HN

R

ab

M-(c+b) M-(e+f) M-(e+f+a) M-(e+f+b)

351 260 243 175

407 316 299 231

from adduct upon CID

long with superimposed fragmentation behavior.

A. Garg et al. / J. Chromatogr. B 879 (2011) 1–7 5

0 5 10 15 20 25 30 35 40

0

20

40

60

80

100 12

3

4

Time (min)

A

12 14 16 18 20 22 24 26 28 30 32

0

20

40

60

80

100

1

23

54

Time (min)

B

150 200 250 300 350 400 450

m/z

0

50

100

404

260

336

243

175

275

292

207

318

361

160

386

421

132

227

440

192

A1

200 300 400

m/z

0

50

100

392

460

348

316

263

477

374

299

429

231

332

179

201

280

B1

200 300 400

0

50

100

290

419

401

376

341

350

205

277

420

333

A2R

elat

ive

Abundan

ceR

elat

ive

Abundan

ce

Rel

ativ

e A

bundan

ceR

elat

ive

Abundan

ceR

elat

ive

Abundan

ce

200 300 400

0

50

100

34

6

43

1

45

7

17

1

41

3

26

1

24

5

19

1

37

1

39

1

29

7

31

7

Rel

ativ

e A

bundan

ce B2

47

5

F -96-Ni 6-NAc

od((a

3m

3

puacnAsmiNtopBtfTp

m/z

ig. 5. TIC chromatograms of targeted MS/MS runs of PQ-NAc (m/z 421) (A) and NPonization modes for PQ-NAc (m/z 421 and 419 respectively) (A1 and A2) and NP-9

f GSH and NAc, the reported fragmentation data [17] were repro-uced, wherein characteristic losses of 75 Da (glycine) and 129 Daglutamic acid) were typical for GSH, while loss of acetyl moiety42 Da) was unique for NAc. The HR-MS data for test compounds (PQnd NP-96) and nucleophiles (GSH and NAc) are given in Table 2.

.2. Identification of covalent adducts of PQ, NAC and theiretabolites

.2.1. GSH adducts with PQ and NP-96Before analyzing the in vitro samples containing test com-

ounds, the viability of microsomal incubation was evaluated bysing acetaminophen as a known substrate. The unique appear-nce of the GSH and NAc adducts (m/z 457 and 313, respectively)onfirmed the enzymatic conversion of acetaminophen to imi-oquinone following by its trapping by the nucleophiles [18].fterwards, the reaction samples of PQ and NP-96 with GSH wereubjected to data dependent mass acquisition to check for the for-ation of adducts. Mass peaks of m/z 565 and 621 gave strong

ndication of the presence of adducts of GSH directly with PQ andP-96, respectively. Subsequently, NLS scans were acquired upon

argeted MS/MS analyses to look for the loss of 129 Da, typicalf GSH adducts [17]. In case of both PQ and NP-96, two isomericeaks of GSH adducts were observed, as shown in Fig. 3A and

, respectively. These were observed in both reaction and con-rol incubations, which showed that both PQ and NP-96 directlyormed adducts with GSH, even without undergoing metabolism.o verify the same, respective MS/MS spectra were recorded inositive and negative ionization modes. As shown in Fig. 3A1, in

m/z

Ac (m/z 477) (B) along with representative MS/MS spectra in positive and negative(m/z 477 and 475 respectively) (B1 and B2).

positive ionization mode, the expected fragment of m/z 436 for PQ-GSH adduct was observed on the loss of 129 Da of glutamic acid.The same 129 Da loss was also observed for NP-96-GSH adduct,with most abundant fragment appearing in this case at m/z 492(Fig. 3B1). In both the cases, fragments pertaining to PQ and NP-96,i.e., m/z 243 + 305, 175 + 305 (Figs. 3A1 and 4), and m/z 299 + 305,m/z 231 + 305 (Figs. 3B1 and 4), respectively, were also observed.Another confirmation came through MS/MS spectra in negativeionization mode (Fig. 3A2 and B2 for PQ and NP-96, respectively),where characteristic fragments of m/z 272 and 254, typical of GSHadducts, were observed [15]. The above was supported by HR-MSdata (Table 3). Further confirmation was provided by justificationof the mass fragmentation pattern for adducts, as shown in Fig. 4.

3.2.2. NAc adducts with PQ and NP-96Similar to the GSH adducts, covalent binding of PQ and NP-96

with NAc were identified in the nucleophile supplemented incuba-tions. Initially, the mass values of the NAc adducts were predictedbased on the already collected GSH adduct data in the positiveionization mode. The values were m/z 421 and 477 for PQ andNP-96, respectively. As neutral loss of 129 Da (typical for GSHadducts) could not happen in NAc in the positive mode, targetedMS/MS analyses for ions of m/z 421(M+H+)/419(M−H−) and m/z477(M+H+)/475(M−H−) were performed for PQ and NP-96, respec-

tively, in both positive and negative modes. The resultant total ionchromatograms (TIC) and representative MS/MS spectra are givenin Fig. 5. As evident, multiple peaks were observed for the predictedmasses in both the reaction and control samples for PQ and NP-96. They produced similar fragmentation pattern, indicating that

6 A. Garg et al. / J. Chromatogr. B 879 (2011) 1–7

Table 3HR-MS data for PQ-GSH, NP-96-GSH, PQ-NAc and NP-96-NAc adducts.

Fragment Experimental mass Best possible molecular formula Exact mass of most possible structure Error in ppm

PQ-GSHM+H+ 565.2413 C25H37N6O7S+ 565.2439 4.599(M+H+)-NH3 548.2171 C25H34N5O7S+ 548.2173 0.364(M+H+)-C5H7NO3 436.2027 C20H30N5O4S+ 436.2013 3.209

NP-96-GSHM+H+ 621.3036 C29H45N6O7S+ 621.3065 4.667(M+H+)-C5H7NO3 492.2681 C24H38N5O4S+ 492.2639 8.532

PQ-NAcM+H+ 421.1885 C20H29N4O4S+ 421.1904 4.511(M+H+)-NH3 404.1620 C20H26N3O4S+ 404.1639 4.701(M+H+)-C5H11N 336.0998 C15H18N3O4S+ 336.1013 4.463

tsNoMt(tttd

3c

9oimwfmwtiamwtls

cinttsmwdp

3o

N

considering that inherently electrophilic compounds are likely tocause toxicity because of their ability to alkylate proteins, DNAand/or GSH. However, the chemical modification should not affectpharmacological efficacy of these compounds.

12 14 16 18 20 22 24 26 28 30 32 34

0

10

20

30

40

50

60

70

80

90

100

12

A

200 300 400 500

m/z

0

50

100

476

408

332

446

247

493

315

367

214

279

185

Rel

ativ

e A

bundan

ce A1

0

50

100

362

447

473

409

329

491

A2

Time (min)

NP-96-NAcM+H+ 477.2529 C24H37N4O4S+

(M+H+)-NH3 460.2252 C24H34N3O4S+

(M+H+)-C5H11N 392.1717 C19H26N3O4S+

hese were isomeric, similar to the observation in GSH. The MS/MSpectra in positive ESI mode (Fig. 5A1 and B1) for both PQ andP-96-NAc adducts showed the presence of characteristic lossesf 17 Da and 85 Da. The same outcome was obtained from targetedS/MS analyses in negative ESI mode, which showed characteris-

ic fragment ions of m/z 290 and m/z 346 in case of PQ and NP-96Fig. 5A2 and B2), respectively. These fragments were formed byhe neutral loss of 2-acetamidoacrylic acid moiety (C5H7NO3). Fur-her, HR-MS studies also confirmed the same (Table 3). Fig. 7 showshe fragmentation of probable adducts, based on collision inducedissociation (CID) data.

.2.3. GSH and NAc adducts of phase I metabolites of the testompounds

The TIC and MS/MS spectra of microsomal incubation of NP-6 (Fig. 6) supplemented with NAc showed two isomeric peaksf m/z 493. As the same were absent in the blank and controlncubations, it indicated covalent adduct formation between the

etabolite and NAc. However, the corresponding GSH adductsere not seen perhaps due to their low intensity in MS. The

ormation of metabolite-NAc adducts was supported by their frag-entation pattern, which was very similar to NP-96-NAc adducts,ith anticipated difference of 16 Da for all the fragments. Also, a

ypical fragment of m/z 362 (Fig. 6A2) was seen in the negative ion-zation mode, similar in line with m/z 346 in case of NP-96-NAcdduct (Fig. 5B2). The probable structure of adduct and its frag-entation pattern are given in Fig. 7. The corresponding adductas not seen in GSH supplemented incubation. It was perhaps due

o the lower analytical sensitivity of GSH adduct of the metabo-ite as the intensity of GSH adduct of the test compounds was alsoignificantly less in LC–MS, as compared to their NAc adduct.

Surprisingly, in the case of microsomal incubations of PQ, weould not detect any reactive metabolites in the sample. Although,n vitro metabolism of PQ is reported elsewhere [19], they have alsoot mentioned formation of hydroxyl metabolites corresponding tohe reactive metabolite of NP-96. Moreover, we could not observehe known carboxy metabolite of PQ, may be due to the lesser sub-trate/protein concentration used in our experiments. Further, toinimize false negatives in this case, efficiency of SPE extractionas validated by comparing the profile from both SPE sample andirect protein precipitation sample. In both cases, the metabolicrofiles were found to be similar.

.2.4. Relevance of the study with respect to the toxicity potentialf PQ and NP-96

In literature, direct covalent binding with nucleophiles (GSH andAc) has been reported for 4-sulfonyl-2-pyridone ring systems [20]

477.2530 0.209460.2265 2.824392.1639 19.889

and more specifically with methoxy 4-O-aryl quinolines [7], similarto our observations in this study with 8-amino substituted quino-lines. This suggests that there may be some role of electrophilicityof PQ with respect to toxicity shown by it. This even means a sim-ilar toxicity potential for NP-96. However, conclusive assessmentwould require relative and quantitative investigation of adduct for-mation potential between the two compounds. These findings maypave the way for further modifications in PQ and NP-96 structures,

200 300 400 500

m/z

Fig. 6. TIC chromatograms of monohydroxy-NP-96-NAc (m/z 493) along with rep-resentative MS/MS spectra in positive (m/z 493) (A1) and negative (m/z 491) (A2)ionization modes.

A. Garg et al. / J. Chromatogr. B 879 (2011) 1–7 7

a

N

OHN

O

S

NH3+

HN

OH

H3CO

bc

d

R

A

N

OHN

O

S

NH3+

HN

OH

H3CO

c

dOH

C(CH3)3

ab

BB

Adducts R M M-a M-b M-c M-(c+a) M-(d+c) M-(d+c+a) M-(c+b) M-(d+c+b)

PQ-NAc H 421 404 336 292 275 260 243 207 175 NP-96-NAc C(CH3)3 477 460 392 348 331 316 299 263 231

Monohydroxy-

NP-96-NAc

- 493 476 408 364 - 332 315 - 247

ents

nohyd

4

(

(

(

A

FRm

[

[[[[

[

[[[

[

Key: M denotes (M+H)+, a-d represents leaving fragm

Fig. 7. Structure of adducts of PQ-NAc and NP-96-NAc (A) and mo

. Conclusions

The following were the significant outcomes of the study:

1) Direct covalent binding of PQ and NP-96 was observed with GSHand NAc. The adducts were formed in all the in vitro incubates,irrespective of the presence of microsomes and NADPH, thoughadducts were intense in microsomal incubates. This direct bind-ing nature of 8-aminoquinoline compounds with nucleophiles,which is being reported for the first time to our knowledge, maybe a possible reason for the toxicity behavior of PQ [21].

2) NP-96 showed liability to be converted to a mono-hydroxymetabolite, which was also observed to form adducts with NAc,similar to its parent.

3) NAc behaved as a better trapping agent for PQ and NP-96 thanGSH perhaps due to its enhanced sensitivity in MS system,though it did not confirm relative tendency of the nucle-ophiles to interact with the compounds involved. Hence, thereis a requirement of the use of established absolute quantita-tive methods [22] to determine differential adduct formationbehavior of PQ and NP-96 with the nucleophiles.

cknowledgements

The authors would like to thank Dr. Amit S. Kalgutkar, Researchellow, Pfizer Global Research and Development, Groton, CT and Dr.amaswami Iyer, Associate Director, Bristol-Myers Squibb, Phar-aceutical Research Institute, Princeton, NJ for helpful discussions.

[

[[

from adduct under CID

roxy-NP-96-NAc (B) with superimposed fragmentation behavior.

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