Identification of the Related Substances in Ampicillin Capsule by ...

Hindawi Publishing Corporation Journal of Analytical Methods in Chemistry Volume 2014, Article ID 397492, 15 pages http://dx.doi.org/10.1155/2014/397492

Research Article Identification of the Related Substances in Ampicillin Capsule by Rapid Resolution Liquid Chromatography Coupled with Electrospray Ionization Tandem Mass Spectrometry Lei Zhang,1 Xian Long Cheng,1,2 Yang Liu,1 Miao Liang,1 Honghuan Dong,1 Beiran Lv,1 Wenning Yang,1 Zhiqiang Luo,1 and Mingmin Tang1 1

School of Chinese Materia Medica, Beijing University of Chinese Medicine, No. 6 Zhonghuan South Road, Wangjing, Chaoyang District, Beijing 100102, China 2 Institute for the Control of Traditional Chinese Medicine and Ethnic Medicine, National Institutes for Food and Drug Control, State Food and Drug Administration, 2 Tiantan Xili, Beijing 100050, China Correspondence should be addressed to Yang Liu; [email protected] Received 15 June 2014; Accepted 8 September 2014; Published 4 November 2014 Academic Editor: Josep Esteve-Romero Copyright © 2014 Lei Zhang et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Rapid Resolution Liquid Chromatography coupled with Electrospray Ionization Tandem Mass Spectrometry (RRLC-ESI-MSn ) was used to separate and identify related substances in ampicillin capsule. The fragmentation behaviors of related substances were used to identify their chemical structures. Finally, a total of 13 related substances in ampicillin capsule were identified, including four identified components for the first time and three groups of isomers on the basis of the exact mass, fragmentation behaviors, retention time, and chemical structures in the literature. This study avoided time-consuming and complex chemosynthesis of related substances of ampicillin and the results could be useful for the quality control of ampicillin capsule to guarantee its safety in clinic. In the meantime, it provided a good example for the rapid identification of chemical structures of related substances of drugs.

1. Introduction Ampicillin is an important semisynthetic 𝛽-lactam antibiotic and it is still widely used nowadays because of its good efficacy in urinary tract infections, respiratory infections, and other diseases caused by germs and bacteria. In recent years, the requirement of quality control for related substances in chemicals became stricter no matter in structure confirmation or content limitation. Ampicillin was especially degradable in presence of aqueous solution or humid storage environment, which would lead to the formation of a variety of degradation products [1]. These related substances (the related substances previously reported were shown in Table 1) would have a great influence on the quality of the products and clinical medication safety. Although there has been much research on the related substances of ampicillin [2, 3], it is not completely explicit so far. To ensure the clinical safety and meet the new requirement of related substances in chemicals [4], it is still necessary

to conduct further studies to develop a rapid and efficient method to describe in more detail the related substances of ampicillin capsule. Many analytical methods including high-performance liquid chromatography (HPLC) [1, 5], high-performance capillary electrophoresis (HPCE) [6], high-performance liquid chromatography-atmospheric pressure chemical ionization mass (HPLC-APCI-MS) [7], and high-performance liquid chromatography-electrospray mass spectrometry (HPLCESI-MS) [8, 9] had been utilized for the analysis of ampicillin. Among these methods, LC-ESI-MS had been shown to be a powerful technique for the analysis of ampicillin and its related substances due to its excellent ability in separation and identification. In this paper, a simple, rapid, and sensitive Rapid Resolution Liquid Chromatography coupled with Electrospray Ionization Tandem Mass Spectrometry (RRLC-ESI-MSn ) method was established for the identification of the related substances in ampicillin capsule. The result suggested that this technique

2

Journal of Analytical Methods in Chemistry

Table 1: The structures of the known related substances of ampicillin. Number

Name of related substances

Chemical structure H

COOH

O N 1

CH3

6-Aminopenicillanic acid (6-APA)

H2 N

CH3

S H

H H

COOH

O N 2

H

L-Ampicillin

CH3

H N

NH2

S

C H

CH3

H

O H

COOH

HN O 3

Diketopiperazines of ampicillin

N H

CH3

H

COOH

OH CH3

HN

H N

Ampicilloic acid

S

O

O

4

CH3

H N

H C

S

CH3

NH2 O H

COOH

O 5

CH3

HN

Ampilloic acid

N H

NH2 H

H

NH2

S H

S

HN 6

H COOH N

Ampicillinyl-D-phenylglycine

O

CH3

O

H O


3

Table 1: Continued. Number


Chemical structure

O H

7

HN

(3R,6R)3,6-Diphenylpiperazine-2,5-dione

NH H O N 8

N

3-Phenylpyrazin-2-ol

OH

H

NH2 O

9

H

D-Phenylglycylampicillin

NH

H

H

H N

CH3 CH3

S N

O

COOH

O H3 C

10

N-Pivaloyl-6-APA

H H

H CH3 CH3

S

HN

H3 C

N

H3 C

COOH

O

O

H

H C 11

N-Pivaloylphenylglycine

COOH CH3

NH

CH3 O

CH3

H 12

D-Phenylglycine

C NH2

COOH

4


Table 1: Continued. Number


Chemical structure

NH2 CH CO NH O 13

CH3

S HN

CH3 COOH NH S CH3 CH CO NH O HN CH3 COOH OH

Open-cycle dimer

NH2 O HN

O 14

Closed-cycle dimer

CH3

S

NH

NH CH CO

CH3 COOH NH O

S N

CH3 CH3 COOH

NH2

15

Open-cycle trimer

CH S CH3 CO NH O HN CH3 COOH NH S CH3 CH CO NH O HN CH3 COOH HN S CH3 CH CO NH O HN CH3 OH COOH NH2

16

Closed-cycle trimer

CH S CH3 CO NH O HN CH3 COOH NH S CH3 CH CO NH O HN CH3 HN COOH S CH3 CH CO NH O N CH3 COOH NH2 CH CO NH O

17

Open-cycle tetramer (𝑛 = 2)

S HN

CH3 CH3 COOH

NH S CH3 CH CO NH CH3 O HN COOH n HN S CH3 CH CO NH O HN CH3 COOH OH


5 Table 1: Continued.

Number

18


Chemical structure NH2 CH S CH3 CO NH CH3 O HN COOH NH S CH3 CH CO NH CH3 O HN COOH n HN S CH3 CH CO NH O N CH3 COOH

Closed-cycle tetramer (𝑛 = 2)

×108

could facilitate rapid and accurate identification of related substances in ampicillin capsule.

2. Experimental

2.2. Preparation of Sample. The contents of ampicillin capsule (equivalent to 10 mg Ampicillin) were dissolved in 10 mL methanol and then filtered through a 0.22 𝜇m syringe filter. And an aliquot (1 𝜇L) of the filtrate was subjected to RRLCESI-MSn for analysis.

0.8 (mAU)

2.1. Chemicals and Materials. Methanol (HPLC grade) was purchased from Fisher Scientific (Pittsburgh, PA, USA). Formic acid (HPLC grade) was obtained from Acros Organics (Geel, Belgium). Deionized water was further purified with a Milli-Q water system (Bedford, Massachusetts, USA). Ampicillin capsule was purchased from DAVA Pharmaceuticals. Inc. (Huntsville, AL, USA). The chromatographic separation was performed with an Agilent 1200 series Rapid Resolution Liquid Chromatography system (Agilent Technologies, USA), equipped with a binary pump, a microvacuum degasser, a high-performance autosampler, a column compartment, a diode array detector, and a MS detector. The samples were separated on a 1.8 𝜇m Agilent Zorbax XDB-C18 column (50 mm × 4.6 mm) at a flow rate of 0.4 mL⋅min−1 . The mobile phases consisted of 0.1% formic acid solution (A) and methanol (B). The optimized RRLC elution conditions were as follows: 0–2 min, 10% B; 2–10 min, 10–20% B; 10–20 min, 20–50% B; 20–25 min, 50% B; 25-25.1 min, 50–10% B; 25.1–30 min, 10% B. DAD spectra were acquired over a scan range of 190–400 nm. The sample volume injected was 1 𝜇L. Agilent 6320 mass spectrometer with an Agilent ChemStation to control and process the data was performed with the ESI source in positive ion mode. The vaporizer temperature was maintained at 300∘ C. The temperature of the drying gas was set at 350∘ C. The flow rate of the drying gas and the pressure of the nebulizing gas were set at 12 L⋅min−1 and 35 psi, respectively. The capillary voltage was kept at 3.5 × 103 V. The mass spectrometer scanned from a mass-to-charge ratio (𝑚/𝑧) 100–900.

15

1.0

10

0.6 7

0.4 3 2 1 4

0.2 0

5

9 6

5

8 10

12 11 1314 15 Time (min)

16

20

25

Figure 1: The total ion chromatography of ampicillin capsule (DAVA). The peaks were numbered according to their retention time.

3. Results and Discussion 3.1. Investigation of the Fragmentation Patterns of Ampicillin. It was necessary to study the characterization of the mass spectra of the parent drug to identify the molecular structure of the related substances in ampicillin capsule. Identifications were based on the fact that the related substances of ampicillin usually contain structural fragments and analogous cleavage characteristic of the parent drug. Structural information and fragmentation mechanisms had been deduced from ions in the mass and collision spectra. This knowledge was useful in the analysis and identification of related substances in ampicillin capsule. We utilized knowledge of characteristic fragment ions of ampicillin and its related substances to identify their structures. Figures 1 and 2 showed the detailed total ion chromatography (TIC) and mass spectrum of ampicillin and its related substances, respectively. Ampicillin yielded an abundant ion in the ESI mass spectrum at 𝑚/𝑧 350.1. The ESI mass spectrum of this ion (𝑚/𝑧 350.1) was shown in Figure 2. The fragment ions at 𝑚/𝑧 106.2 and 160.0 were reported to arise from the benzylamine group and the thiazolidine ring. The fragment ion at 𝑚/𝑧 192.0 was

m/z

500

450

400

500

400

450

500

450

400

800

700

600

+MS, 19.3 min

Peak 16

2 445.1

4

800

700

600

699.2

500

400

100

200

246.0 328.3

300

158.0

m/z +MS2 (525.0), 19.3 min

6 Intens.

500

400 524.8

571.2

730.3 889.3

160.0

2

m/z

Figure 2: The mass spectrum of 16 chemicals in ampicillin capsule.

m/z

817.3

800

700

673.2

600

500

300

200

400

407.1488.8

0 100

800

700

682.2

600

500

483.0

400

300

m/z

×105

540.1

248.0

300

100 Intens.

m/z

531.1

389.2

3

800

700

600

400

500

300

200

100

160.1

0

+MS2 (699.7), 17.0 min

200

199.0

1

596.2

(548.5), 13.9 min

3 2

×106 4

699.1

+MS, 17.0 min

381.1

350

443.1

+MS 358.1

200

Intens. m/z

454.4

600

m/z

0

540.1

500

400

300

100

80 100 120 140 160 180 200 220 240 260 280

200

158.0 199.0

1

1.0

+MS, 13.9 min

Peak 12

4

106.1 119.0

Peak 15

300

250

200

150

100

m/z

2

+MS (208.8), 12.8 min

5 4 3 2 1 0

339.0

358.1

2

256.2

366.9

225.9 207.9

×105

190.9

350.1

300

+MS2 (384.7), 10.0 min

548.1

0

135.0

350

m/z

4 2

224.1237.7

175.1

158.0

300

250

200

100

150

Intens. Intens.

Intens.

158.0

429.1 413.2

279.1 328.3

160.0

114.1 140.0

500

450

400

350

300

135.1

350

250

200

100 150 200 250 300 350 400 450 500

150

100

450

400

500 500

450

400

350

300

200

250 250

200

384.9

159.0 190.9

6

80 100 120 140 160 180 200 220 240 260 280 Intens. Intens.

466.0 439.1

158.0 130.1 107.1

×106

m/z

×105 3.0 2.5 2.0 1.5 1.0 0.5 0.0

800

700

600

500

350

300

250

10 15 20 25 30 35 40 45 50 150

100 100

150

Intens. 800

700

600

500

400

400

200

100 Intens. Intens.

500

450

400

300

250

200

300

m/z

150

450

400

500 500

450

400

350

300

250

200

150

Intens.

0.0

107.1

Peak 8

0.0

191.0

0.5

+MS, 10.0 min

0.5

319.1

+MS, 12.8 min Peak 11

×106

2

+MS (483.5), 15.5 min

307.1

279.1

1.0

208.1

0.0

324.1350.1 160.0 221.9 189.0

365.1

m/z

1.0

0.5

m/z 239.1 267.0

1.0

100

800

700

600

429.2

+MS2 (324.4), 2.8 min

1.5

333.1

259.0

106.2 160.0

1.5

673.2 597.3 699.2

1.5

0.5

639.3 584.1

500

400

300

200

100

0.00

429.1

178.1 206.0

100

514.2 217.0

337.3

600

158.0

130.1

400

Intens.

673.2

130.1

0.4

×105

+MS, 15.2 min

1.00

0.6

Peak 14

100

600

500

400

337.3

+MS, 15.5 min

0.0

m/z

×106 1.25

2

×106

483.1

0.2

750.4 840.3

700

655.1

381.0

300

100

200

191.0

m/z

0.8

800

324.1

160.0

300

Peak 13

324.1350.1 465.9 307.1 439.1

×106 1.0

514.1

+MS2 (673.0), 15.2 min

1.25 1.00 0.75 0.50 0.25 0.00

+MS (483.4), 12.3 min

500

m/z

×105

2

100 150 200 250 300 350 400 450 500 550

800

700

600

400

500

362.1

300

200

100

0.0

239.1 267.0

300

1.0

+MS (382.3), 9.0 min

0.0

m/z

×106 5 4 3 2 1 0

307.0

384.0

×105 2.0

2

3

114.0

376.0 514.0 413.7

253.0

m/z 223.0

1.5

Intens.

Intens.

1.5 191.0 248.1

130.1

100

2

+MS (566.5), 11.1 min

0.5

0.5

800

700

600

500

400

300

200

100

407.1

2.0

1.0

0.0

m/z

×106

0.25

100

407.0 350.1

157.9 107.1

159.9 130.0 107.1

×106

+MS, 12.3 min

Peak 10

200

2

1.5

+MS, 9.0 min

0.5

0 m/z

200

4

191.9

483.1

m/z

m/z

×106 1.50 1.25 1.00 0.75 0.50 0.25 0.00

382.1

1.0

1

×107

+MS, 11.1 min

Intens.

566.1

333.0 305.0

Peak 7

10 15 20 25 30 35 40 45 50

m/z Peak 9

0

0

500

450

400

350

250

200

150

217.9

192.0

×106

+MS2 (364.8), 8.1 min

2 106.2 128.0

174.0

0.5

0.0

m/z

3 1

333.1 288.9

300

192.0

6

0.50

Intens.

500

450

174.0

1.0

1.5

173.9 214.9 328.2 391.1429.2 195.0237.0278.1 350.1 480.6

4 Intens.

106.2

+MS, 8.1 min

158.0

174.0

+MS (350.3), 2.5 min

106.1

×107

Peak 6

107.1 130.0

2

m/z

364.1

×105

+MS2 (350.2), 4.2 min

×106 Intens.

400

350

300

250

m/z 159.9

100

Intens.

200

100

150

159.9 191.9 106.1

1.0 0.8 0.6 0.4 0.2 0.0

0.75

100

500

0.4

×107

Intens.

450

0.6

Intens.

Intens.

Peak 5

0.0

Intens.

+MS, 4.2 min

m/z 160.0

0.0

m/z

×106 1.50 1.25 1.00 0.75 0.50 0.25 0.00

107.1 158.9 130.1

1.5

175.1 279.2 106.1147.1 209.1

200

350.1

0.8

0.2

Intens.

400

350

m/z

×108 1.0

307.2

1 0

300

200

100

250

209.1 279.2 106.2 175.0 147.2

0

2

150

Intens.

307.1

150

Intens.

4 2

+MS (368.4), 2.2 min

3

×106 1.0 0.8 0.6 175.0 0.4 147.1 201.0 0.2 106.1 128.1 0.0

Peak 3

×106

2

350

+MS (368.7), 1.8 min

6

324.1

×106 324.1 +MS, 2.8 min 1.0 Peak 4 0.8 0.6 130.1 158.0 0.4 107.1 368.1 175.1 0.2 307.1 396.1 195.0 224.0 279.0 429.1 0.0

+MS, 2.5 min

4 0

m/z

×106

324.1 2

350

307.1351.1

350.1

6 2

m/z

×105

Intens.

324.2

300

100

500

450

350

300

400

391.3 429.3

283.2

Peak 2

250

324.2

×106 8

368.1 +MS, 2.2 min

200

Intens.

Peak 1

×106 5 4 3 2 1 130.1 107.1 158.1 0 150

368.1 +MS, 1.8 min

250

200

150

100

Intens.

×106 1.2 1.0 0.8 0.6 107.1 158.0 0.4 130.1 192.0 0.2 224.1 0.0

Intens.


Intens.

6


7 O H N

O

H CH

+

C

NH

O

N

CH3

+O

H

H

H

+

O H

CH3

S

+

O

H m/z 192.0

H2 O H

O

O

C

m/z 174.0

∙∙

+

H

O N H C

CH3

H N

+

CH3

S H

H m/z 333.1

O −NH3

+

H

COOH

O

+

N H

NH2

H N

CH3

S

C H O

CH3

H

C H

NH

m/z 106.1

Ampicillin m/z 350.1

+

H

COOH

N

CH3 S

CH3

m/z 160.0

Figure 3: Proposed fragmentation pathways and characteristic ions of protonated ampicillin (𝑚/𝑧 350).

proposed to arise as a result of losing a −NH2 group at the benzylamine side chain followed by an oxygen rearrangement and cleavage of the 𝛽-lactam ring. The fragment ion at 𝑚/𝑧 174.0 could be attributed to the loss of H2 O from the fragment at 𝑚/𝑧 192.0, but it might arise from other pathways. The proposed fragmentation pathways of ampicillin were shown in Figure 3. 3.2. Identification of the Known Related Substances in Ampicillin Capsule. This part of the investigation focused on the characterization of the ESI-MS properties of the parent

drug and its known related substances. Table 2 showed the chromatographic and mass spectral characteristics of the detected related substances in ampicillin capsule. Peak 1 and Peak 2 showed the same MS data. All of them produced protonated quasimolecular ion at 𝑚/𝑧 368.1 [M + H]+ , major ions at 𝑚/𝑧 324.1, 307.1, 279.2, and 175.1 in ESI+ mode. Based on diagnostic ions (𝑚/𝑧 324.1, 307.1, and 175.1) and comparison with the published literature of known related substances of ampicillin [10], Peak 1 and Peak 2 were identified as isomers of ampicilloic acid. (5S, 6R) or (5R, 6R) ampicilloic acids were the two groups of ampicilloic

8

Journal of Analytical Methods in Chemistry +

H

COOH

HN

H N

CH3

H C

CH3

S

+

H

NH2 O m/z 324.1

+

H O +

NH2

O

NH2

m/z 151.0

O

HN

HN

H C

CH3

N

COOH

OH

H N

COOH

CH3

S

CH3 CH3

S

NH2 O m/z 368.1

m/z 307.1 +

H H2 C

N S

COOH

HN

CH3 CH3

+

H

COOH

H2 C

NH2 m/z 279.1

CH3 S

CH3

m/z 175.0

Figure 4: Proposed fragmentation pathway for the fragmentation ions of ampicilloic acid (𝑚/𝑧 368).

Table 2: Results of identification of the known related substances in ampicillin capsule. Peak number 1 2 3 4 5 6 7 10 15 16

RT (min) 1.8 2.2 2.5 2.8 4.2 8.1 9.0 12.3 17.0 19.3

MS (𝑚/𝑧) 368.1 [M + H]+ 368.1 [M + H]+ 350.1 [M + H]+ 324.1 [M + H]+ 350.1 [M + H]+ 364.1 [M + K+ H]+ 382.1 [M + MeOH]+ 483.1 [M + H]+ 699.1 [M + H]+ 524.8 [1/2M + H]+

MS2 (𝑚/𝑧) 324.1; 307.1; 279.2 324.1; 307.2; 279.2; 175.1 333.0; 192.0; 174.0; 160.0; 106.1 307.0; 279.1; 201.0; 175.0; 147.1; 128.1; 106.1 333.1; 192.0; 174.0; 159.9; 106.2 191.9; 174.0; 128.0; 106.2 331.1; 223.0; 206.0; 160.0; 106.2 439.1; 350.0; 267.0; 239.1 540.1; 381.1; 248.0 889.3; 730.3; 571.2; 160.0

acid isomers which were reported to be the metabolites and degradation products of ampicillin [1]. According to the retention behavior in reversed-phase chromatography of Peak 1 and Peak 2 and the related literature [1], Peak 1 and Peak 2 were tentatively identified as (5S, 6R) ampicilloic acid and (5R, 6R) ampicilloic acid, respectively. Figure 4 showed the proposed MS fragmentation pathway for the fragmentation ions of ampicilloic acid. Peak 3 produced a protonated molecular ion at 𝑚/𝑧 350.1 [M + H]+ , fragment ions at 𝑚/𝑧 333.0 [M − NH3 ]+ , 192.0, 174.0, 160.0, and 106.1 in ESI+ mode. Peak 5 was clearly identified as ampicillin based on comparison of its retention time and mass spectrometric data with reference standards

Identification (5S, 6R) ampicilloic acid (5R, 6R) ampicilloic acid L-Ampicillin (5S) or (5R) ampilloic acids Ampicillin (5S) or (5R) ampilloic acids Diketopiperazines of ampicillin D-Phenylglycylampicillin Closed-cycle dimer Closed-cycle trimer

[8]. Peak 3 showed the same fragment ions, fragmentation pattern, and characteristic ions as Peak 5. Therefore, we could conclude that Peak 3 was an isomer of Peak 5. Considering that Peak 3 had a much shorter retention time than Peak 5, and with the comparison of related substances reported in the literature [1], Peak 3 was tentatively identified as L-ampicillin. Figure 5 showed the proposed MS fragmentation pathway for the fragmentation ions of L-ampicillin. Peak 4 gave a protonated molecular ion [M + H]+ with an 𝑚/𝑧 value of 324.1, major fragment ions at 𝑚/𝑧 307.0, 279.1, 201.0, 175.0, 147.1, 128.1, and 106.1 in ESI+ mode. Peak 4 was tentatively identified as (5R) or (5S) ampilloic acid based on its characteristic ions (𝑚/𝑧 324.1, 307.0, 279.1, 128.1, and


9 O

O

H CH

+

C

NH

O

N

CH3

+O

H

H

H

+

O H

CH3

S

+

H N

O

H m/z 192.0 H2 O

H

O

O

C

m/z 174.0

∙∙

+

H

O N H C

CH3

H N

+

H

H m/z 333.0

O −NH3

+

H

COOH

O

+

N H

NH2

H N

CH3 CH3

S

C H O

CH3

S

C H

NH

m/z 106.1

H

L-Ampicillin m/z 350.1

+

H

COOH

N

CH3 S

CH3

m/z 160.0

Figure 5: Proposed fragmentation pathways and characteristic ions of protonated L-ampicillin (𝑚/𝑧 350).

106.1) and comparison with the published literature of known related substances of ampicillin [10]. Peak 6 produced major fragment ions at 𝑚/𝑧 364.1 [M + K + H]+ , 191.9, 174.0, 128.0, and 106.2. They all had similar MS fragmentation patterns (𝑚/𝑧 191.9, 174.0, 128.0, and 106.2). By comparison with the published literature [1], Peak 6 was tentatively identified as (5R) or (5S) ampilloic acid. (5S) or (5R) ampilloic acids were the isomers of ampilloic acids which were reported to be metabolites or degradation products of ampicillin [1]. However, the exact structure of these two components could

not be determined due to the limited information. Figure 6 showed the proposed MS fragmentation pathway for the fragmentation ions of ampilloic acids. Peak 7 had a molecular weight of 350 ([M + MeOH+ H]+ , 𝑚/𝑧 382.1) and five major fragment ions were observed at 𝑚/𝑧 331.1, 223.0 [191 + MeOH]+ , 206.0 [174 + MeOH]+ , 160.0, and 106.2. As it was reported [8], the two characteristic fragment ions 𝑚/𝑧 160.0 and 106.2 were the representative fragment ions of ampicillin. By comparison with the published literature [7], this component was tentatively identified

10

Journal of Analytical Methods in Chemistry +

HN

CH3 CH3 S m/z 128.1

H2 C

+

+

H

COOH

S

+

COOH

O HN

CH3

HN

H2 C

H

CH3

m/z 175.0

CH3 S Ampilloic acid (m/z 324.1)

N H

NH2

+

−CO

COOH

H O

NH

CH3

m/z 106.1

m/z 279.1

CH3

N

CH3

HN

S m/z 307.0

Figure 6: Proposed fragmentation pathway for the fragmentation ions of ampilloic acids.

H N

O

CH2

N H

−H2 O

O

m/z 206.0[174 + MeOH]+

m/z 223.0[191 + MeOH]+

H

COOH

HN O

H N S

NH

H

CH3 CH3

COOH

N

CH3 S

m/z 106.2

N H

O

CH3

m/z 160.0

m/z 382.1[M + MeOH]+

Figure 7: Proposed fragmentation pathway for the fragmentation ion of diketopiperazines of ampicillin.

as diketopiperazines of ampicillin. Figure 7 showed the proposed MS fragmentation pathway for the fragmentation ion of diketopiperazines of ampicillin. Peak 10 produced a protonated molecular ion at 𝑚/𝑧 483.1 [M + H]+ , the major fragment ions at 𝑚/𝑧 439.1, 350.0, 267.0, and 239.1 in ESI+ mode. Fragment ion at 𝑚/𝑧 439.1 could be attributed to loss of one −COOH from the ion (𝑚/𝑧

483.1). Based on the mass spectra, Peak 10 was identified as Dphenylglycylampicillin [1, 11]. Figure 8 showed the proposed MS fragmentation pathway for the fragmentation ion of Dphenylglycylampicillin. Peak 15 had a molecular weight of 𝑚/𝑧 699.1 [M + H]+ and three major fragment ions 𝑚/𝑧 540.1, 381.1, and 248.0 in ESI+ mode. Upon collision-induced dissociation (CID),


11

NH2

H

H

+

H

CH3

S

H N

CH3 N

O

+

COOH

O

H

O

m/z 350.0

H HN

H

NH2

NH

O m/z 438.1

−COOH

+

H

H

NH

H S

H N

CH3 CH3

N

O O

H O m/z 267.0

COOH

H

D-Phenylglycylampicillin (m/z 483.1)

H

+

H CH3

S

NH2

CH3 N

COOH

O

H +

[M + Na] m/z 239.1

Figure 8: Proposed fragmentation pathway for the fragmentation ion of D-phenylglycylampicillin.

Table 3: Results of identification of the unknown related substances in ampicillin capsule. Peak number 9 12 13 14

RT (min) 11.1 13.9 15.2 15.5

MS (𝑚/𝑧) 566.1 [M + H]+ 548.1 [M + H]+ 673.2 [M + H]+ 483.1 [M + H]+

MS2 (𝑚/𝑧) 407.1; 248.1; 191.0 443.1; 358.1; 199.0 655.1; 514.1; 324.1; 191.0 439.1; 350.1; 239.1; 160.0

the ion at 𝑚/𝑧 699.1 eliminated one molecule of thiazolidine ring to produce 𝑚/𝑧 540.1. The 𝑚/𝑧 540.1 ion could further lose one molecule of thiazolidine ring successively to give significant 𝑚/𝑧 381.1 fragment ion. Thus, Peak 15 was identified as closed-cycle dimer based on the published literature [1]. Closed-cycle dimer was the main cause of allergy, so that we must control the amount of this related substance in ampicillin capsule. Peak 16 produced major fragment molecular ions at 𝑚/𝑧 524.8 [M + H]+ , 889.3, 730.3, 571.2, and 160.0 in ESI+ mode. Upon CID, the ion at 𝑚/𝑧 1048 [M]+ eliminated one molecule of thiazolidine ring to produce 𝑚/𝑧 889.3. The 𝑚/𝑧 889.3 ion could lose one molecule of thiazolidine ring successively to give significant 𝑚/𝑧 730.3 ion. The 𝑚/𝑧 730.3 ion could further lose one molecule of thiazolidine ring successively to give significant 𝑚/𝑧 571.2. The fragment ion 𝑚/𝑧 160.0 is characteristic thiazolidine ring of ampicillin. Thus, Peak 16

Identification Ampicilloic acid and 6-APA oligomer 6-APA ampicillin amide Ampilloic acids and ampicilloic acids oligomer Isomer of D-phenylglycyl ampicillin

was identified as closed-cycle trimer based on the published literature [1]. Closed-cycle trimer was also the main cause of allergy, so that we must control the amount of this related substance in ampicillin capsule. 3.3. Identification of the Unknown Related Substances in Ampicillin Capsule. This part of the investigation was to identify the chemical structures of unknown related substances which were not yet reported in ampicillin capsule based on the mass fragment characterization and cleavage pathways of ampicillin and its known related substances. By means of the RRLC-ESI-MSn experiments, in this part, chemical structures of four related substances were tentatively identified in ampicillin capsule for the first time. Table 3 showed the chromatographic and mass spectral characteristics of the above unknown related substances detected by RRLC-ESIMSn in ampicillin capsule.

12

Journal of Analytical Methods in Chemistry NH +

COOH

H

O O HN

H N

CH3

H C NH2 O

H H3 C

CH3

S m/z 407.1

H

+

NH

S

NH

+

O H3 C

N

O

COOH

H

O

O HOOC

H

HN

H N

H N

CH3

H C

S

CH3

H C NH2 O

NH2 O

m/z 248.1

Ampicilloic acid and 6-APA oligomer m/z 566.1

+

O

H N

N H

O

m/z 191.0

Figure 9: Proposed fragmentation pathway for the fragmentation ion of ampicilloic acid and 6-APA oligomer.

Peak 9 had a molecular weight of 𝑚/𝑧 566.1 [M + H]+ and three major fragment ions 𝑚/𝑧 407.1, 248.1, and 191.0 in ESI+ mode. The fragment ions at 𝑚/𝑧 407.1 and 248.1 eliminated one molecule of thiazolidine ring successively from ion at 𝑚/𝑧 566.1 [M + 1]+ . The fragment ion at 𝑚/𝑧 191.0 probably should be a characteristic fragment ion of ampicillin piperazine-2,5-dione [12]. Thus, Peak 9 was identified tentatively as ampicilloic acid and 6-aminopenicillanic acid (6-APA) oligomer. Figure 9 showed the proposed MS fragmentation pathway for the fragmentation ion of ampicilloic acid and 6-APA oligomer. Peak 12 produced a protonated molecular ion at 𝑚/𝑧 548.1 [M + H]+ and three major fragment ions at 443.1, 358.1, and 199.0. Based on fragment ions, Peak 12 was tentatively identified as 6-APA ampicillin amide. Figure 10 showed the

proposed MS fragmentation pathway for the fragmentation ion of 6-APA ampicillin amide. Peak 13 had a molecular weight of 673.2 [M + H]+ and four major fragment ions 𝑚/𝑧 655.1, 514.1, 324.1, and 191.0. The fragment ions at 𝑚/𝑧 655.1 and 514.1 were attributed to the loss of one water molecule (18 Da) and one molecule of thiazolidine ring from ion at 𝑚/𝑧 673.2. The fragment ion at 𝑚/𝑧 324.1 was molecular weight of ampilloic acids. The fragment ion at 𝑚/𝑧 191.0 was the fragment ion of ampicilloic acids. Peak 13 was identified tentatively as ampilloic acids and ampicilloic acids oligomer. Figure 11 showed the proposed MS fragmentation pathway for the fragmentation ion of ampilloic acids and ampicilloic acids oligomer. Peak 14 produced a protonated molecular ion at 𝑚/𝑧 483.1 [M + H]+ , which was identified as the other isomer of


13 +

COOH

H

O

CH3

N CO

H

N H

N

H

CH3

H

m/z 358.1

CH3

S

CH3

S

+

H

COOH

H

O

CH3

N CO

H

O

N H

N H

CH3

H N

NH2

H

H

H

CH3

S

C

CH3

S

H

O 6-APA ampicillin amide m/z 548.1

+

+

COOH

H

O

N CO

H

O

CH3

N N

S H

H

N H

CH3

S H

O

CO

H

CH3

N

CH3

H S

CH3

m/z 443.0

NH

H

H

CH3

m/z 198.9

O

Figure 10: Proposed fragmentation pathway for the fragmentation ion of 6-APA ampicillin amide.

D-phenylglycylampicillin because Peak 14 and Peak 10 both showed the same diagnostic ions at 𝑚/𝑧 439.1, 350.1, 239.1, and 160.0.

4. Conclusion The RRLC-ESI-MSn technique was successfully established to rapidly determine and identify the structures of the related substances in ampicillin capsule. RRLC is efficient in separating chemical compounds in a mixture, and MS provides abundant information for structural elucidation of the compounds when tandem mass spectrometry is applied [13]. Although ampilloic acids, ampicilloic acid, and closedcycle dimer had been investigated previously by LC-MS method, MS information and characteristic diagnostic ions of

a number of components in ampicillin capsule were described simultaneously for the first time. None of the previously reported methods have led to so much chemical information on the related substances in ampicillin capsule. The results of this study had identified 13 out of 15 related substances in ampicillin capsule. Unfortunately, three groups of isomers (Peak 1 and Peak 2, Peak 4 and Peak 6, and Peak 10 and Peak 14) and condensation of amino and carboxyl groups (Peak 9, Peak 12, and Peak 13) could not be identified fully by current RRLC-ESI-MSn information. Peak 8 and Peak 11 had not yet been identified based on current mass spectra information. In summary, this investigation had provided an example of the rapid identification of related substances in ampicillin capsule. The meaningful information for the related substances in ampicillin capsule could lead to the development of the understanding of the quality and safety of the drug.

14

Journal of Analytical Methods in Chemistry H H

H

H N

NH2

S

C

CH3 CH3

HN O

O

CO

OH H

H

H N

NH C

m/z 514.1

H H

NH2

S

CH3 CH3

HN O

O

O

H

H N

C

CH3

CO

OH H

H

S

NH

NH HN

C O

CH3 CH3 COOH

H Ampilloic acids and ampicilloic acids oligomer m/z 673.2

O NH2

H

S

NH

C

CH3 CH3

HN O m/z 324.1

H N

N H

O

COOH

H m/z 191.0

Figure 11: Proposed fragmentation pathway for the fragmentation ion of ampilloic acids and ampicilloic acids oligomer.

Conflict of Interests The authors declare that there is no conflict of interests.

Authors’ Contribution

by NMR,” Journal of Pharmaceutical and Biomedical Analysis, vol. 30, no. 4, pp. 1075–1085, 2002. [3] O. Shakoor and R. B. Taylor, “Analysis of ampicillin, cloxacillin and their related substances in capsules, syrups and suspensions by high-performance liquid chromatography,” Analyst, vol. 121, no. 10, pp. 1473–1477, 1996.

Lei Zhang and Xian Long Cheng contributed equally in this work.

[4] EMA, “Guideline on setting specifications for related impurities in antibiotics,” http://www.ema.europa.eu/docs/en GB/document library/Scientific guideline/2012/07/WC500129997.pdf.

References

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[6] C. Q. Niu and S. Q. Zhu, “Separation and determination of ampicillin polymers by high performance capillary electrophoresis,” Acta Pharmaceutica Sinica, vol. 32, no. 3, pp. 207–209, 1997.

Journal of Analytical Methods in Chemistry [7] S. Horimoto, T. Mayumi, K. Aoe, N. Nishimura, and T. Sato, “Analysis of 𝛽-lactam antibiotics by high performance liquid chromatography-atmospheric pressure chemical ionization mass spectrometry using bromoform,” Journal of Pharmaceutical and Biomedical Analysis, vol. 30, no. 4, pp. 1093–1102, 2002. [8] R. F. Straub and R. D. Voyksner, “Determination of penicillin G, ampicillin, amoxicillin, cloxacillin and cephapirin by highperformance liquid chromatography—electrospray mass spectrometry,” Journal of Chromatography, vol. 647, no. 1, pp. 167–181, 1993. [9] E. Verdon, R. Fuselier, D. Hurtaud-Pessel, P. Cou¨edor, N. Cadieu, and M. Laurentie, “Stability of penicillin antibiotic residues in meat during storage ampicillin,” Journal of Chromatography A, vol. 882, no. 1-2, pp. 135–143, 2000. [10] S. Suwanrumpha and R. B. Freas, “Identification of metabolites of ampicillin using liquid chromatography/thermospray mass spectrometry and fast atom bombardment tandem mass spectrometry,” Biomedical and Environmental Mass Spectrometry, vol. 18, no. 11, pp. 983–994, 1989. [11] B. P. Commission and G. Britain, British Pharmacopoeia 2011, Stationery Office, 2010. [12] S. Suwanrumpha, D. A. Flory, R. B. Freas, and M. L. Vestal, “Tandem mass spectrometric studies of the fragmentation of penicillins and their metabolites,” Biomedical and Environmental Mass Spectrometry, vol. 16, no. 1-12, pp. 381–386, 1988. [13] M. Ye, J. Han, H. Chen, J. Zheng, and D. Guo, “Analysis of phenolic compounds in rhubarbs using liquid chromatography coupled with electrospray ionization mass spectrometry,” Journal of the American Society for Mass Spectrometry, vol. 18, no. 1, pp. 82–91, 2007.

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