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P. Zou et al., Eur. J. Mass Spectrom. 13, 233–238 (2007)

233

Letter: Liquid chromatography ion-trap time-of-flight mass spectrometric study on the fragmentation of an acetildenafil analogue

Peng Zou,a,b Sharon Sze-Yin Oh,b Kin-Har Kiang,b Min-Yong Lowb and Hwee-Ling Kohc,* Centre for Analytical Science, Health Sciences Authority, 11 Outram Road, Singapore 169078

a

Division of Pharmaceutics, College of Pharmacy, Ohio State University, 357 Parks Hall, 500W 12th Ave, Columbus, OH 43210, USA

b

Centre for Analytical Science, Health Sciences Authority, 11 Outram Road, Singapore 169078

c

Department of Pharmacy, National University of Singapore, 18 Science Drive 4, Singapore 117543. E-mail: [email protected]

Liquid chromatography ion-trap time-of-flight mass spectrometry was employed to elucidate the fragmentation pathways of an analogue of acetildenafil. Based on the accurate masses of the parent ion, product ions and neutral losses of acetildenafil analogue, its fragmentation pathways were proposed. The information is useful for the on-line structural identification of unknown analogues of acetildenafil found as adulterants in herbal products. Keywords: acetildenafil analogue, fragmentation pathway, LC/IT/ToF-MS, herbal product

Acetildenafil (Figure 1) was synthesised as a phospho diesterase-5 (PDE-5) inhibitor for the treatment of erectile dysfunction.1 In the past several years, acetildenafil and its analogues have been found to be adulterated into herbal products and dietary supplements which are advertised as “all natural”.2–6 It is potentially dangerous for patients to unknowingly consume herbal products and dietary supplements adulterated with acetildenafil or its analogues. Hence, identification of acetildenafil-related compounds in herbal products is important and urgent. Recent studies have demonstrated the advantages of multi-stage mass spectrometry (MSn) and accurate mass spectrometry in structural characterisation of synthetic adulterants in herbal complexes.7–9 Online identification of unknown modified analogues of acetildenafil will be possible if the characteristic fragmentation pathways of this class of compounds have been elucidated. To date, the fragmentation pathways of two other PDE-5 inhibitors, sildenafil

DOI: 10.1255/ejms.873

21

O

20

6 HN

O 19

5

18

14

17

15 16

O

10 7

8

N1 N2

N 4

9

3 11

13

22 23 25

N

26

29 28

N 27 R

R=CH3 Acetildenafil; R=H Analogue Figure 1. Chemical structures of acetildenafil and its analogue.

ISSN 1469-0667

© IM Publications 2007

234

Study of the Fragmention of an Acetildenafil Analogue

and tadalafil, have been described.10–12 Gratz et al. proposed possible product ion structures for two analogues of acetildenafil using accurate mass measurements through Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS).8 Liquid chromatography ion-trap time-of-fight mass spectrometry (LC/IT/ToF-MS) is a powerful tool for fragmentation study because of its capacity for MSn analysis and accurate mass measurement of parent ions, product ions and neutral losses. In this paper, we report the elucidation of fragmentation pathways of an analogue of acetildenafil using LC/IT/ToF-MS. Acetildenafil and its analogue were isolated from two herbal products, respectively, and their structures were characterised using nuclear magnetic resonance and mass spectrometry.3 As shown in Figure 1, the analogue is structurally similar to acetildenafil except that the N-ethylpiperazine group in acetildenafil has been replaced by an N-methylpiperazine group. Low resolution tandem mass sectrometry (MS/MS) analysis was performed on a triplequadrupole API 2000 mass spectrometer coupled with an electrospray ionization source (Applied Biosystems,

Ontario, Canada). Acetildenafil and its analogue were individually dissolved in acetonitrile / H2O (1 : 1, v / v) at a concentration of 1 µg mL–1. The solutions were infused into the spectrometer at a flow rate of 3 µL min–1 using an external syringe pump. The [M + H] + was selected as the precursor ion to generate ESI/MS/MS spectra. Collision energy (CE) was set at 50. Data acquisition and processing were conducted using Analyst software. The MS n scan and accurate measurement were performed on a liquid chromatography hybrid ion-trap and time-of-flight system coupled with an ESI interface (Shimadzu Corporation, Nakagyo-ku, Japan). Mass spectrometry parameters were set as: positive ESI; scan range m/z 50–1200; probe voltage 4.5 kV; CDL temperature 250°C; block heater temperature 200°C; nebulising gas 1.5 L min–1 of nitrogen; ion accumulation time 50 msec; CID energy MS2 35%, MS3 60% and MS4 75%; collision gas MS/MS 50%, MS3 70% and MS4 80%. 5 µL of the acetildenafil analogue solution (1 µg mL–1) was injected onto a Shim-pack VP-ODS (2 mm × 150 mm). The column oven temperature was 40°C. The isocratic elution profile was 30% of water and 70% of acetonitrile

Acetildenafil

Analogue

Figure 2. Low resolution MS/MS spectra of acetildenafil and its analogue

Figure 2. Low resolution MS/MS spectra of acetildenafil and its analogue.


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Figure 3. Typical accurate MSn spectra of protonated analogue of acetildenafil (m/z 453 → m/z 353 → m/z 325 → m/z 297).

(v / v), maintained for 15 min. The flow rate of the mobile phase was 0.2 mL min–1. Six replicate accurate mass measurements were conducted to generate an average value. Elemental compositions of the analogue’s product ions and neutral losses were predicted using Composition Formula Predictor (Shimadzu Corporation, Nakagyo-ku, Japan). Very wide tolerance restrictions were set up: (a) C, H, N, O, S and P (minimum number is 0 and maximum numbers are 50, 300, 12, 5 and 5, respectively), (b) apply nitrogen rule and (c) error margin varied from ±1 to ±100 ppm.

Figure 2 shows the low resolution MS/MS spectra of acetildenafil and its analogue. The [M + H]+ and three product ions of the analogue (m/z 453, 113 and 97) have a mass shift of 14 Da compared to corresponding product ions of acetildenafil, suggesting that the product ions at m/z 113 and 97 contained the methyl group on the piperazine. The product ions at m/z 396, 353, 341, 325, 311, 297 and 166 were detected in the MS/MS spectra of both acetildenafil and its analogue, suggesting that the ethyl or methyl group on the piperazine was eliminated.

325 ĺ m/z 297)

MS stage Observed ions Neutral loss Observed ions Neutral loss Observed ions

MS

MS/MS

453.2610

453.2610 100.1015

(C3H7N)

(C5H12N2) 353.1595

MS4

325.1289

297.1362

27.9927 (CO)

325.1289

112.1016 (C6H12N2) 341.1594

28.0306 (C2H4) MS3

453.2610

453.2610

57.0587 396.2023

Neutral loss Observed ions Neutral loss Observed ions

453.2610 142.1111 (C7H14N2O)

311.1499

113.1081 [C6H13N2]+

159.0398 (C8H5N3O)

166.0891 [C9H12NO2]+

Scheme 1. Fragmentation pathways of protonated acetildenafil elemental formulas Scheme 1. Fragmentation pathways of protonated acetildenafil analogue andanalogue elementaland formulas of neutral losses. of

neutral

236


Table 2. Accurate mass data of the protonated acetildenafil analogue and its product ions (n = 6).

Table 1. Accurate mass data of neutral losses (n = 6).

Formula

Theoretical

Found

Error (ppm)

C8H5N3O

159.0433

159.0398

–22.0

C7H14N2O

142.1106

142.1111

3.5

C6H12N2

112.1001

112.1016

13.4

C5H12N2

100.1001

100.1015

14.0

C3H7N

57.0579

57.0587

14.0

C2H4

28.0313

28.0306

24.9

CO

27.9949

27.9927

–78.5

Formula

LC/IT/ToF-MS was employed to measure the accurate mass of the protonated analogue and it was determined as O N

O HN

Theoretical

Found

Error (ppm)

C24H33N6O3

453.2609

453.2610

0.2

C21H26N5O3

396.2030

396.2023

–1.8

C19H21N4O3

353.1608

353.1595

–3.7

C18H21N4O3

341.1608

341.1594

–4.1

C17H17N4O3

325.1295

325.1289

–1.8

C17H19N4O2

311.1503

311.1499

–1.3

C16H17N4O2

297.1346

297.1362

5.4

C9H12NO2

166.0863

166.0891

16.8

C6H13N2

113.1073

113.1081

7.1

O

O N

N

O HN

N +H+

+H+

O HN N

N

HO

N

N

N

N

N

m/z 113.1081

m/z 453.2610

m/z 453.2610 C5H12N2

C3H7N

C6H12N2

O N

O HN

N

N

N

O

N

O HN

N

O N

O HN

N

N

O

O N

O HN

N

N

O

C7H14N2O

N

N

O

N

O HN N

HO

N m/z 341.1594

m/z 353.1595

m/z 396.2023

CH2=CH2 O N

OH HN

N

N C 8H 5N 3O OH

O

CO

NH3 m/z 325.1289

O m/z 166.0891

N

OH HN

N

N O m/z 297.1362

Scheme 2. Tentatively proposed structures of product ions of protonated acetildenafil analogue.

m/z 311.1499

N


453.2610 (average value, n = 6). The accurate mass was used to predict the ion’s elemental formula. 40, 19, 13 and 10 candidate formulae were obtained when the error margin was set as 10, 5, 3 and 2 ppm, respectively. There were still five candidates even though the error margin was set as 1 ppm. Typical mass accuracy of a ToF-MS is 2–5 ppm. Hence, it is difficult to directly determine the elemental formula of an ion with a high or medium m/z based on ToF mass measurements. Figure 3 shows the typical MS n spectra of the protonated analogue of acetildenafil (m/z 453 → m/z 353 → m/z 325 → m/z 297). The protonated analogue underwent multiple stage fragmentation and produced small neutral molecules. The accurate masses of these neutral losses were deduced from the mass differences between parent ions and product ions. The elemental formulae of small neutral molecules were easily determined because the number of candidate formulae was limited. As shown in Scheme 1 and Table 1, the elemental formulae of all the small neutral losses were unambiguously determined. CO was the only candidate formula for the neutral loss 27.9927, even though the error margin was set as 100 ppm. For neutral loss 159.0398, the first candidate was C3H5N5O3 (theoretical mass 159.0392, error 3.8 ppm) but the formula was not chemically rational. The second candidate, C8H5N3O, was determined as the elemental formula of the neutral loss. Then, the elemental formulae of product ions were deduced from the elemental formulae of the corresponding parent ions and neutral losses. The elemental formula of the product ion at m/z 113.1081 was determined directly from its m/z. The elemental formulae of product ions and mass errors are shown in Table 2. Based on the elemental formulae of the protonated analogue, its product ions and neutral losses, the structures of product ions were tentatively proposed in Scheme 2. The product ion at m/z 113 was expected to contain the methylpiperazine group and derived from the protonated analogue by cleavage of the bond between C22 and C23. Product ions at m/z 396, 353, 341 and 311 were observed to be produced from protonated analogue and methylpiperazine group was partly or completely eliminated. Among them, the product ion at m/z 311 was also observed in the MS/MS spectra of protonated sildenafil.10 The ion at m/z 353 generated product ions at m/z 325, 297 and 166 by sequential neutral losses. The product ion at m/z 297 was observed to be derived from ion m/z 353 (Figure 3) instead of protonated acetildenafil analogue as reported.8 This study elucidates the fragmentation pathways of a protonated analogue of acetildenafil using LC/IT/ToF-MS and tentatively proposes the structures of its product ions. The results suggest that the MSn and accurate mass measurement capabilities of LC/IT/ToF-MS make it a powerful tool for fragmentation study. The data reported in this paper is useful for the detection and structural determination of modified analogues of acetildenafil found as adulterants in herbal products.

237

Acknowledgement The technical assistance by Dr Zhaoqi Zhan from Shimadzu Pte Ltd is acknowledged. References 1.

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Received: 24 June 2007 Revised: 27 June 2007 Accepted: 27 June 2007 Publication: 30 August 2007