Research Article Identification of the Related Substances ...

Research ArticleIdentification of the Related Substances in AmpicillinCapsule by Rapid Resolution Liquid Chromatography Coupledwith 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 14 October 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 wereused to identify their chemical structures. Finally, a total of 13 related substances in ampicillin capsule were identified, includingfour 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 relatedsubstances 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 antibioticand it is still widely usednowadays because of its good efficacyin urinary tract infections, respiratory infections, and otherdiseases caused by germs and bacteria. In recent years, therequirement of quality control for related substances in chem-icals became stricter no matter in structure confirmation orcontent limitation. Ampicillin was especially degradable inpresence of aqueous solution or humid storage environment,which would lead to the formation of a variety of degradationproducts [1]. These related substances (the related substancespreviously reported were shown in Table 1) would have agreat influence on the quality of the products and clinicalmedication safety.

Although there has been much research on the relatedsubstances of ampicillin [2, 3], it is not completely explicit sofar. To ensure the clinical safety and meet the new require-ment of related substances in chemicals [4], it is still necessary

to conduct further studies to develop a rapid and efficientmethod to describe in more detail the related substances ofampicillin capsule.

Many analytical methods including high-performanceliquid chromatography (HPLC) [1, 5], high-performance cap-illary electrophoresis (HPCE) [6], high-performance liquidchromatography-atmospheric pressure chemical ionizationmass (HPLC-APCI-MS) [7], and high-performance liquidchromatography-electrospray mass spectrometry (HPLC-ESI-MS) [8, 9] had been utilized for the analysis of ampicillin.Among these methods, LC-ESI-MS had been shown to bea powerful technique for the analysis of ampicillin and itsrelated substances due to its excellent ability in separation andidentification.

In this paper, a simple, rapid, and sensitive Rapid Resolu-tion Liquid Chromatography coupled with Electrospray Ioni-zationTandemMass Spectrometry (RRLC-ESI-MSn)methodwas established for the identification of the related substancesin ampicillin capsule.The result suggested that this technique

Hindawi Publishing CorporationJournal of Analytical Methods in ChemistryVolume 2014, Article ID 397492, 15 pageshttp://dx.doi.org/10.1155/2014/397492

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

1 6-Aminopenicillanic acid (6-APA)N

S

H

HH

O COOH

CH3

CH3H2N

2 L-Ampicillin

N

S

H H

HO

C

O

HN

H

COOH

CH3

CH3

NH2

3 Diketopiperazines of ampicillin

NH

HNO

O

S

H COOH

CH3

CH3

HN

4 Ampicilloic acidS

HO

HN

HC

O

HN

COOH

CH3

CH3

NH2

OH

5 Ampilloic acid

SNH

OH COOH

NH2

HN CH3

CH3

6 Ampicillinyl-D-phenylglycine N

SHH

O HO

HH

O

NH2

HNCOOH

Journal of Analytical Methods in Chemistry 3

Table 1: Continued.


7 (3R,6R)3,6-Diphenylpiperazine-2,5-dione

H

H

O

O

NH

HN

8 3-Phenylpyrazin-2-ol

N

N

OH

9 D-Phenylglycylampicillin

N

S

HO

H H

H

H

O

O

HN

NHCH3

CH3

COOH

NH2

10 N-Pivaloyl-6-APA N

S

HO

H H

O

H3C

H3C

H3C

HN

COOH

CH3

CH3

11 N-PivaloylphenylglycineC

H

O

NH

COOHCH3

CH3

CH3

12 D-Phenylglycine C

H

COOH

NH2


Table 1: Continued.


13 Open-cycle dimer

S

O

S

O

CH

CH

CH3

CH3

CH3

CH3

COOH

COOH

NH2

CO

CO

NH

NHNH

HN

HNOH

14 Closed-cycle dimer

S

N

S

O

O

O

NH

NHNH

CH3

CH3

COOH

CH3

CH3

COOH

NH2

HN

CH CO

15 Open-cycle trimer

HNOH

S

O

S

O

S

O

CH3

CH3COOH

CH3

CH3COOH

CH3

CH3COOH

NH2

NH

NHNH

NH

HN

HNHN

CH

CH

CH

CO

CO

CO

16 Closed-cycle trimer

N

S

O

S

O

S

O

CH3

CH3COOH

CH3

CH3COOH

CH3

CH3COOH

NH2

NH

NHNH

NH

HN

HNHN

CH

CH

CH

CO

CO

CO

17 Open-cycle tetramer (𝑛 = 2) S

ONH

NHCH

HNHN

COOH

CH3

CH3

CO

n

COOH

CH3

CH3

OH

S

ONHCH

HNCO

S

ONH

CHNH2

HNCOOH

CH3

CH3

CO


Table 1: Continued.


18 Closed-cycle tetramer (𝑛 = 2)

S

O

S

O

N

S

O

NH

NHNH

NH

CH

CH

CH

NH2

HN

HNHN

COOH

CH3

CH3

COOH

CH3

CH3

COOH

CH3

CH3

CO

CO

CO

n

could facilitate rapid and accurate identification of relatedsubstances in ampicillin capsule.

2. Experimental

2.1. Chemicals and Materials. Methanol (HPLC grade) waspurchased from Fisher Scientific (Pittsburgh, PA, USA).Formic acid (HPLC grade) was obtained from Acros Organ-ics (Geel, Belgium). Deionized water was further purifiedwith a Milli-Q water system (Bedford, Massachusetts, USA).Ampicillin capsule was purchased from DAVA Pharmaceuti-cals. Inc. (Huntsville, AL, USA).

The chromatographic separation was performed with anAgilent 1200 series Rapid Resolution Liquid Chromatogra-phy system (Agilent Technologies, USA), equipped with abinary pump, a microvacuum degasser, a high-performanceautosampler, a column compartment, a diode array detector,and a MS detector. The samples were separated on a 1.8 𝜇mAgilent Zorbax XDB-C

18column (50mm× 4.6mm) at a flow

rate of 0.4mL⋅min−1. The mobile phases consisted of 0.1%formic acid solution (A) and methanol (B). The optimizedRRLC elution conditions were as follows: 0–2min, 10% B;2–10min, 10–20% B; 10–20min, 20–50% B; 20–25min, 50%B; 25-25.1min, 50–10% B; 25.1–30min, 10% B. DAD spectrawere acquired over a scan range of 190–400 nm. The samplevolume injected was 1 𝜇L. Agilent 6320 mass spectrometerwith an Agilent ChemStation to control and process the datawas performed with the ESI source in positive ion mode.The vaporizer temperature was maintained at 300∘C. Thetemperature of the drying gas was set at 350∘C. The flow rateof the drying gas and the pressure of the nebulizing gas wereset at 12 L⋅min−1 and 35 psi, respectively.The capillary voltagewas kept at 3.5 × 103 V.The mass spectrometer scanned froma mass-to-charge ratio (𝑚/𝑧) 100–900.

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

0 5 10 15 20 25

0.2

0.4

0.6

0.8

1.0

4

5

6

7

8

9

10

11

12

1314

15

16

123

(mAU

)

Time (min)

×108

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

3. Results and Discussion

3.1. Investigation of the Fragmentation Patterns of Ampicillin.It was necessary to study the characterization of the massspectra of the parent drug to identify the molecular structureof the related substances in ampicillin capsule. Identificationswere based on the fact that the related substances of ampicillinusually contain structural fragments and analogous cleavagecharacteristic of the parent drug. Structural information andfragmentation mechanisms had been deduced from ions inthe mass and collision spectra. This knowledge was usefulin the analysis and identification of related substances inampicillin capsule. We utilized knowledge of characteristicfragment ions of ampicillin and its related substances toidentify their structures. Figures 1 and 2 showed the detailedtotal ion chromatography (TIC) and mass spectrum ofampicillin and its related substances, respectively.

Ampicillin yielded an abundant ion in the ESI mass spec-trum at 𝑚/𝑧 350.1. The ESI mass spectrum of this ion (𝑚/𝑧350.1) was shown in Figure 2.The fragment ions at𝑚/𝑧 106.2and 160.0 were reported to arise from the benzylamine groupand the thiazolidine ring. The fragment ion at𝑚/𝑧 192.0 was


130.1158.0

192.0224.1 283.2

324.2

391.3429.3

0.0

0.2

0.4

0.6

0.8

1.0

1.2

100

150

200

250

300

350

400

450

500

106.2147.2

175.0209.1

279.2

307.1

324.1

0

2

4

6

100

150

200

250

300

350

400

450

500

107.1130.1

158.1 307.1351.1

368.1368.1

324.2

0

1

2

3

4

5

100

150

200

250

300

350

400

450

500

106.1147.1175.1

209.1279.2

307.2

324.1

0

1

2

3

107.1130.1

350.1

158.9

0

2

4

6

8

100

150

200

250

300

350

400

450

500

10

15

20

25

30

35

40

45

50

107.1158.0

175.1195.0

224.0 279.0307.1

368.1396.1

429.1

324.1

130.1

0.0

0.2

0.4

0.6

0.8

1.0

100

150

200

250

300

350

400

450

500

106.1

128.1

147.1175.0

201.0

279.1

307.0

0.0

0.2

0.4

0.6

0.8

1.0

100

150

200

250

300

350

400

450

500

107.1

m/z

m/z

m/z

m/z

m/z

m/z

m/z

m/z

m/z

m/z

m/z

m/z m/z

m/zm/z

m/z

m/z

m/zm/z

m/z

m/z

m/z

m/z

m/z

m/z

m/z

m/z

m/z

m/z

m/z

m/z

m/z

+MS, 1.8min

Peak 1 Peak 2 Peak 3 Peak 4

×106

×106

×106

×106

×106

×106

×106

×106

Inte

ns.

Inte

ns.

Inte

ns.

Inte

ns.

Inte

ns.

Inte

ns.

Inte

ns.

Inte

ns.

Inte

ns.

Inte

ns.

Inte

ns.

Inte

ns.

Inte

ns.

Inte

ns.

Inte

ns.

Inte

ns.

Inte

ns.

Inte

ns.

Inte

ns.

Inte

ns.

+MS, 2.2min +MS, 2.5min +MS, 2.8min

100

150

200

250

300

350

400

450

500

106.1

159.9191.9

350.1

0.0

0.2

0.4

0.6

0.8

1.0

100

150

200

250

300

350

400

450

500

106.2

159.9

174.0

192.0

217.9 288.9333.1

0.0

0.2

0.4

0.6

0.8

1.0

100

150

200

250

300

350

400

450

500

107.1130.0

173.9

195.0214.9

237.0278.1

328.2350.1

391.1429.2480.6

364.1

158.0

0.00

0.25

0.50

0.75

1.00

1.25

1.50

100

150

200

250

300

350

400

450

500

0

1

2

3

4

10

15

20

25

30

35

40

45

50

107.1130.0

382.1

159.9

0.0

0.5

1.0

1.5

0.0

0.5

1.0

1.5

100

150

200

250

300

350

400

450

500

106.2160.0

178.1

206.0

223.0

259.0319.1

333.1

365.1

0

1

2

3100

150

200

250

300

350

400

450

500

107.1130.1

158.0

190.9 279.1 328.3

384.0

413.2429.1

384.9

159.0

0.00

0.25

0.50

0.75

1.00

1.25

1.50

100

150

200

250

300

350

400

450

500

114.1140.0

160.0

207.9225.9

339.0

366.9

0.0

0.5

1.0

1.5

2.0

100

150

200

250

300

350

400

450

500

107.1157.9 350.1

566.1

407.0

0

2

4

6

100

200

300

400

500

600

700

800

191.0248.1

362.1

407.1

0.0

0.5

1.0

1.5

2.0

100

200

300

400

500

600

700

800

130.1 253.0376.0

413.7 514.0

483.1

0.0

0.5

1.0

1.5

100

200

300

400

500

600

700

800

160.0

239.1

267.0

307.1

324.1350.1

439.1465.9

0

1

2

3

4

5

100

150

200

250

300

350

400

450

500

550

107.1

114.0

135.1

158.0

175.1 224.1237.7

208.1

191.0

0.0

0.5

1.0

1.5

80

100

120

140

160

180

200

220

240

260

280

106.1119.0

135.0

190.9

0.0

0.5

1.0

1.5

2.0

2.5

3.0

80

100

120

140

160

180

200

220

240

260

280

158.0199.0

358.1

548.1

0

2

4

6

100

200

300

400

500

600

700

800

0

1

2

3

4400

500

600

191.0

324.1

381.0

514.1

655.1750.4 840.3

0.00

0.25

0.50

0.75

1.00

1.25

100

200

300

400

500

600

700

800

130.1

158.0

217.0 429.1

514.2

584.1639.3

337.3

673.2

0.00

0.25

0.50

0.75

1.00

1.25

100

200

300

400

500

600

700

800

337.3429.2

597.3

673.2

699.2

483.1

130.1

0.0

0.2

0.4

0.6

0.8

1.0

100

200

300

400

500

600

700

800

160.0189.0221.9

239.1267.0

307.1

324.1350.1

439.1466.0

0.0

0.5

1.0

1.5

100

200

300

400

500

600

256.2

350.1

454.4

540.1

596.2

699.1

158.0

0.0

0.5

1.0

1.5

100

200

300

400

500

600

700

800

248.0

381.1

483.0

540.1

682.20

1

2

3

4

5

100

200

300

400

500

600

700

800

246.0328.3445.1 699.2

524.8

158.0

0

1

2

3

4

100

200

300

300

400

500

600

700

800

160.0

407.1488.8

571.2

673.2

730.3

817.3

889.3

0

2

4

6

100

200

300

400

500

600

700

800

+MS2(368.7), 1.8min×10

5

×105 ×10

5

×105

×105

×105 ×10

5

×105

+MS2(368.4), 2.2min +MS2(350.3), 2.5min +MS2(324.4), 2.8min

+MS, 4.2min×10

8

Peak 5

+MS, 8.1min×10

6

×106

Inte

ns.

Inte

ns.

Inte

ns.

Inte

ns.

Inte

ns.

Inte

ns.

Inte

ns.

Inte

ns.

Inte

ns.

Inte

ns.

Inte

ns.

Inte

ns.

×106

×106

×106

×106 ×10

6

×106

×106

×106

×106

×106

Peak 6+MS, 9.0min

×107

×107

×107

Peak 7+MS, 10.0min

Peak 8

+MS2(350.2), 4.2min +MS2(364.8), 8.1min+MS2(382.3), 9.0min +MS2(384.7), 10.0min

+MS, 11.1min

Peak 9

+MS, 12.3minPeak 10

+MS, 12.8minPeak 11

+MS, 13.9minPeak 12

+MS2(566.5), 11.1min +MS2(483.4), 12.3min +MS2(208.8), 12.8min +MS2(548.5), 13.9min

160.1

199.0

358.1

389.2

443.1

531.1

100

200

Peak 13

+MS2(673.0), 15.2min

+MS, 15.2min

+MS, 15.5min

Peak 14

+MS2(483.5), 15.5min

+MS, 17.0min

Peak 15

+MS2(699.7), 17.0min

+MS, 19.3minPeak 16

+MS2(525.0), 19.3min

106.1

160.0

174.0

192.0

305.0333.0

106.2128.0

174.0

191.9

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


N

S

H H

HO

C

O

HN

H CH

OH

HNO

O

H

N

S

H

N

S

H H

HCO

C

O

HN

O

O

H

N

S

H H

CO

O

H

H

NH

NH

CH

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

+

+

+

+

+

+

+

+

O

H2O

−NH3

COOH

COOH

NH2

∙∙

m/z 174.0

m/z 192.0

m/z 333.1

m/z 106.1

m/z 160.0

Ampicillin m/z 350.1

H+

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

proposed to arise as a result of losing a −NH2group at the

benzylamine side chain followed by an oxygen rearrangementand cleavage of the 𝛽-lactam ring. The fragment ion at 𝑚/𝑧174.0 could be attributed to the loss ofH

2O from the fragment

at 𝑚/𝑧 192.0, but it might arise from other pathways. Theproposed fragmentation pathways of ampicillin were shownin Figure 3.

3.2. Identification of the Known Related Substances in Ampi-cillin Capsule. This part of the investigation focused onthe characterization of the ESI-MS properties of the parent

drug and its known related substances. Table 2 showed thechromatographic and mass spectral characteristics of thedetected related substances in ampicillin capsule.

Peak 1 and Peak 2 showed the same MS data. All ofthem produced protonated quasimolecular ion at 𝑚/𝑧 368.1[M +H]+, major ions at 𝑚/𝑧 324.1, 307.1, 279.2, and 175.1in ESI+ mode. Based on diagnostic ions (𝑚/𝑧 324.1, 307.1,and 175.1) and comparison with the published literature ofknown related substances of ampicillin [10], Peak 1 and Peak2 were identified as isomers of ampicilloic acid. (5S, 6R) or(5R, 6R) ampicilloic acids were the two groups of ampicilloic


S

HO

HN

HC

O

N

S

HO

S

H

O

S

H

HN

HC

O

N

S

H

C

++

+

+

+

+

COOH COOH

COOH

COOH

COOH

HN

HNHN

HN

OH

H2C

H2

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

NH2

NH2

NH2

NH2

NH2

m/z 324.1

m/z 368.1m/z 151.0

m/z 279.1

m/z 175.0

m/z 307.1

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 RT (min) MS (𝑚/𝑧) MS2 (𝑚/𝑧) Identification1 1.8 368.1 [M + H]+ 324.1; 307.1; 279.2 (5S, 6R) ampicilloic acid2 2.2 368.1 [M + H]+ 324.1; 307.2; 279.2; 175.1 (5R, 6R) ampicilloic acid3 2.5 350.1 [M + H]+ 333.0; 192.0; 174.0; 160.0; 106.1 L-Ampicillin4 2.8 324.1 [M + H]+ 307.0; 279.1; 201.0; 175.0; 147.1; 128.1; 106.1 (5S) or (5R) ampilloic acids5 4.2 350.1 [M + H]+ 333.1; 192.0; 174.0; 159.9; 106.2 Ampicillin6 8.1 364.1 [M + K+H]+ 191.9; 174.0; 128.0; 106.2 (5S) or (5R) ampilloic acids7 9.0 382.1 [M + MeOH]+ 331.1; 223.0; 206.0; 160.0; 106.2 Diketopiperazines of ampicillin10 12.3 483.1 [M + H]+ 439.1; 350.0; 267.0; 239.1 D-Phenylglycylampicillin15 17.0 699.1 [M + H]+ 540.1; 381.1; 248.0 Closed-cycle dimer16 19.3 524.8 [1/2M + H]+ 889.3; 730.3; 571.2; 160.0 Closed-cycle trimer

acid isomers which were reported to be the metabolitesand degradation products of ampicillin [1]. According tothe retention behavior in reversed-phase chromatography ofPeak 1 andPeak 2 and the related literature [1], Peak 1 andPeak2 were tentatively identified as (5S, 6R) ampicilloic acid and(5R, 6R) ampicilloic acid, respectively. Figure 4 showed theproposed MS fragmentation pathway for the fragmentationions of ampicilloic acid.

Peak 3 produced a protonated molecular ion at𝑚/𝑧 350.1[M +H]+, fragment ions at 𝑚/𝑧 333.0 [M −NH

3]+, 192.0,

174.0, 160.0, and 106.1 in ESI+ mode. Peak 5 was clearlyidentified as ampicillin based on comparison of its retentiontime and mass spectrometric data with reference standards

[8]. Peak 3 showed the same fragment ions, fragmentationpattern, and characteristic ions as Peak 5.Therefore, we couldconclude that Peak 3 was an isomer of Peak 5. Consideringthat Peak 3 had a much shorter retention time than Peak 5,andwith the comparison of related substances reported in theliterature [1], Peak 3 was tentatively identified as L-ampicillin.Figure 5 showed the proposedMS fragmentation pathway forthe fragmentation ions of L-ampicillin.

Peak 4 gave a protonated molecular ion [M +H]+ withan 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 4was tentatively identified as (5R) or (5S) ampilloic acid basedon its characteristic ions (𝑚/𝑧 324.1, 307.0, 279.1, 128.1, and


NH

+

+

+

N

SH H

HO

C

O

HN

H CH

OH

HNO

O

H

N

S

H

N

SH H

HCO

C

O

HN

O

O

H

N

SH H

C

O

O

O

H

H

H2O

NH

CH

+

+

+

+

+

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

NH2

m/z 174.0

m/z 192.0

m/z 106.1

m/z 160.0

m/z

∙∙

333.0−NH3

COOH

COOH

L-Ampicillin m/z 350.1

H+

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

106.1) and comparison with the published literature of knownrelated substances of ampicillin [10]. Peak 6 produced majorfragment 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 thepublished literature [1], Peak 6 was tentatively identified as(5R) or (5S) ampilloic acid. (5S) or (5R) ampilloic acidswere the isomers of ampilloic acids which were reportedto 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 6showed the proposed MS fragmentation pathway for thefragmentation 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 observedat 𝑚/𝑧 331.1, 223.0 [191 +MeOH]+, 206.0 [174 +MeOH]+,160.0, and 106.2. As it was reported [8], the two characteristicfragment ions 𝑚/𝑧 160.0 and 106.2 were the representativefragment ions of ampicillin. By comparison with the pub-lished literature [7], this component was tentatively identified


NH

+

+

+

+

+

HN

H2C

CH3

CH3

m/z 106.1

m/z 279.1

m/z 175.0

COOH

SNH

OH

S

H

S

N

S

HO

m/z 128.1

H2C

CH3

CH3

CH3

CH3

HN HN

NH2

COOH

CH3

CH3

COOH

HN

−CO

m/z 307.0

Ampilloic acid (m/z 324.1)

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

NH

CH3

CH3

CH3

CH3

m/z 160.0

COOH

COOH

NH

HNO

O

S

H

S

N

H

NH

HNO

O

m/z 223.0[191 + MeOH]+

−H2O

HN

m/z 382.1[M + MeOH]+

m/z 106.2

CH2

m/z 206.0[174 + MeOH]+

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

as diketopiperazines of ampicillin. Figure 7 showed the pro-posed MS fragmentation pathway for the fragmentation ionof diketopiperazines of ampicillin.

Peak 10 produced a protonated molecular ion at 𝑚/𝑧483.1 [M +H]+, the major fragment ions at𝑚/𝑧 439.1, 350.0,267.0, and 239.1 in ESI+ mode. Fragment ion at 𝑚/𝑧 439.1could be attributed to loss of one −COOH from the ion (𝑚/𝑧

483.1). Based on themass spectra, Peak 10was identified asD-phenylglycylampicillin [1, 11]. Figure 8 showed the proposedMS fragmentation pathway for the fragmentation ion of D-phenylglycylampicillin.

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 248.0in ESI+ mode. Upon collision-induced dissociation (CID),


+

+

+

+

HN

NH2

NH2

NH2

N

S

HO

H H

H

H

O

O

HN

N

S

HO

H HH

O

HN

H

H

O

O

N

S

HO

H HCH3

CH3

CH3

CH3

CH3

CH3

COOH

NH

NH

m/z 267.0

m/z 350.0

−COOH

COOH

COOH

m/z 438.1

[M + Na]+ m/z 239.1

D-Phenylglycylampicillin (m/z 483.1)

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 RT (min) MS (𝑚/𝑧) MS2 (𝑚/𝑧) Identification9 11.1 566.1 [M + H]+ 407.1; 248.1; 191.0 Ampicilloic acid and 6-APA oligomer12 13.9 548.1 [M + H]+ 443.1; 358.1; 199.0 6-APA ampicillin amide13 15.2 673.2 [M + H]+ 655.1; 514.1; 324.1; 191.0 Ampilloic acids and ampicilloic acids oligomer14 15.5 483.1 [M + H]+ 439.1; 350.1; 239.1; 160.0 Isomer of D-phenylglycyl ampicillin

the ion at 𝑚/𝑧 699.1 eliminated one molecule of thiazolidinering to produce 𝑚/𝑧 540.1. The 𝑚/𝑧 540.1 ion could furtherlose one molecule of thiazolidine ring successively to givesignificant 𝑚/𝑧 381.1 fragment ion. Thus, Peak 15 was identi-fied as closed-cycle dimer based on the published literature[1]. Closed-cycle dimer was the main cause of allergy, sothat we must control the amount of this related substance inampicillin capsule.

Peak 16 produced major fragment molecular ions at 𝑚/𝑧524.8 [M +H]+, 889.3, 730.3, 571.2, and 160.0 in ESI+ mode.UponCID, the ion at𝑚/𝑧 1048 [M]+ eliminated onemoleculeof thiazolidine ring to produce 𝑚/𝑧 889.3. The 𝑚/𝑧 889.3ion could lose one molecule of thiazolidine ring successivelyto give significant 𝑚/𝑧 730.3 ion. The 𝑚/𝑧 730.3 ion couldfurther lose one molecule of thiazolidine ring successivelyto give significant 𝑚/𝑧 571.2. The fragment ion 𝑚/𝑧 160.0 ischaracteristic thiazolidine ring of ampicillin. Thus, Peak 16

was identified as closed-cycle trimer based on the publishedliterature [1]. Closed-cycle trimer was also the main cause ofallergy, so that we must control the amount of this relatedsubstance in ampicillin capsule.

3.3. Identification of the UnknownRelated Substances in Ampi-cillin Capsule. This part of the investigation was to identifythe chemical structures of unknown related substances whichwere not yet reported in ampicillin capsule based on themass fragment characterization and cleavage pathways ofampicillin and its known related substances. By means ofthe RRLC-ESI-MSn experiments, in this part, chemical struc-tures of four related substances were tentatively identifiedin ampicillin capsule for the first time. Table 3 showed thechromatographic and mass spectral characteristics of theabove unknown related substances detected by RRLC-ESI-MSn in ampicillin capsule.


+

+

+

+

HN

HN

NH2

NH2

NH2

CH3

CH3

NHNH

COOH

COOH

S

H

HN

HC

O

N

S

HO

HH

O

S

H

HN

HC

O

OO

HN

HC

O

OO

NH

HNO

O

NH

m/z 248.1

m/z 407.1

m/z 191.0

Ampicilloic acid and 6-APA oligomer m/z 566.1

HOOC

H3C

H3C

CH3

CH3

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 inESI+ mode. The fragment ions at 𝑚/𝑧 407.1 and 248.1 elim-inated one molecule of thiazolidine ring successively fromion at 𝑚/𝑧 566.1 [M + 1]+. The fragment ion at 𝑚/𝑧 191.0probably should be a characteristic fragment ion of ampi-cillin piperazine-2,5-dione [12]. Thus, Peak 9 was identifiedtentatively as ampicilloic acid and 6-aminopenicillanic acid(6-APA) oligomer. Figure 9 showed the proposed MS frag-mentation pathway for the fragmentation ion of ampicilloicacid and 6-APA oligomer.

Peak 12 produced a protonated molecular ion at 𝑚/𝑧548.1 [M +H]+ and three major fragment ions at 443.1, 358.1,and 199.0. Based on fragment ions, Peak 12 was tentativelyidentified as 6-APA ampicillin amide. Figure 10 showed the

proposed MS fragmentation pathway for the fragmentationion of 6-APA ampicillin amide.

Peak 13 had a molecular weight of 673.2 [M +H]+ andfour 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 tothe loss of one water molecule (18Da) and one molecule ofthiazolidine ring from ion at 𝑚/𝑧 673.2. The fragment ionat 𝑚/𝑧 324.1 was molecular weight of ampilloic acids. Thefragment ion at𝑚/𝑧 191.0 was the fragment ion of ampicilloicacids. Peak 13 was identified tentatively as ampilloic acids andampicilloic acids oligomer. Figure 11 showed the proposedMS fragmentation pathway for the fragmentation ion ofampilloic acids and ampicilloic acids oligomer.

Peak 14 produced a protonated molecular ion at 𝑚/𝑧483.1 [M +H]+, which was identified as the other isomer of


N

S

HN

C

H

O

O

H H

H

N

SNH

O

H H

H

N

SN

O

O

H H

H

N

SNH

O

H H

H

N

S

H

H

N

SNH

O

H H

H

N

S

H

H

O

H

+

+

+

+

NH2

CH3

CH3

CH3

CH3

NH

COOH

COOH

COOH

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

m/z 358.1

m/z 198.9m/z 443.0

CO

CO

CO

CO

6-APA ampicillin amide m/z 548.1

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

D-phenylglycylampicillin because Peak 14 and Peak 10 bothshowed 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 establishedto rapidly determine and identify the structures of therelated substances in ampicillin capsule. RRLC is efficientin separating chemical compounds in a mixture, and MSprovides abundant information for structural elucidation ofthe compounds when tandem mass spectrometry is applied[13]. Although ampilloic acids, ampicilloic acid, and closed-cycle dimer had been investigated previously by LC-MSmethod,MS information and characteristic diagnostic ions of

a number of components in ampicillin capsulewere describedsimultaneously for the first time. None of the previouslyreported methods have led to so much chemical informationon the related substances in ampicillin capsule. The resultsof this study had identified 13 out of 15 related substancesin ampicillin capsule. Unfortunately, three groups of isomers(Peak 1 and Peak 2, Peak 4 and Peak 6, and Peak 10 and Peak14) and condensation of amino and carboxyl groups (Peak 9,Peak 12, and Peak 13) could not be identified fully by currentRRLC-ESI-MSn information. Peak 8 and Peak 11 had not yetbeen identified based on currentmass spectra information. Insummary, this investigation had provided an example of therapid identification of related substances in ampicillin cap-sule. The meaningful information for the related substancesin ampicillin capsule could lead to the development of theunderstanding of the quality and safety of the drug.


HN

HN

HN

HN

OH

OH

NH2

NH2

NH2

NH

NH NH

NH

COOH

COOH

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CO

CO

SHN

C

H H

H

O

H

O

S

C

H

H

O

S

C

H

H

O

SHN

C

H H

H

O

H

O

HN

C

H

O

NH

HNO

O

Ampilloic acids and ampicilloic acids oligomer m/z 673.2

m/z 514.1

m/z 324.1

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

Lei Zhang and Xian Long Cheng contributed equally in thiswork.

References

[1] Y. Zhu, E. Roets, Z. Ni, M. L. Moreno, E. Porqueras, and J.Hoogmartens, “Evaluation of liquid chromatography methodsfor the separation of ampicillin and its related substances,”Journal of Pharmaceutical and Biomedical Analysis, vol. 14, no.5, pp. 631–639, 1996.

[2] M. Shamsipur, Z. Talebpour, H. R. Bijanzadeh, and S.Tabatabaei, “Monitoring of ampicillin and its related substances

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, cloxacillinand their related substances in capsules, syrups and suspensionsby high-performance liquid chromatography,” Analyst, vol. 121,no. 10, pp. 1473–1477, 1996.

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

[5] C. Larsen and H. Bundgaard, “Polymerization of penicillins.V. Separation, identification and quantitative determination ofantigenic polymerization products in ampicillin sodium prep-arations by high performance liquid chromatography,” Journalof Chromatography, vol. 147, pp. 143–150, 1978.

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


[7] S. Horimoto, T. Mayumi, K. Aoe, N. Nishimura, and T. Sato,“Analysis of 𝛽-lactam antibiotics by high performance liq-uid chromatography-atmospheric pressure chemical ionizationmass spectrometry using bromoform,” Journal of Pharmaceuti-cal and Biomedical Analysis, vol. 30, no. 4, pp. 1093–1102, 2002.

[8] R. F. Straub and R. D. Voyksner, “Determination of penicillinG, ampicillin, amoxicillin, cloxacillin and cephapirin by high-performance liquid chromatography—electrospray mass spec-trometry,” Journal of Chromatography, vol. 647, no. 1, pp. 167–181,1993.

[9] E. Verdon, R. Fuselier, D. Hurtaud-Pessel, P. Couedor, N.Cadieu, and M. Laurentie, “Stability of penicillin antibiotic res-idues in meat during storage ampicillin,” Journal of Chromatog-raphy A, vol. 882, no. 1-2, pp. 135–143, 2000.

[10] S. Suwanrumpha and R. B. Freas, “Identification of metabolitesof ampicillin using liquid chromatography/thermospray massspectrometry and fast atom bombardment tandem mass spec-trometry,” 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 ofpenicillins and their metabolites,” Biomedical and Environmen-tal Mass Spectrometry, vol. 16, no. 1-12, pp. 381–386, 1988.

[13] M. Ye, J. Han, H. Chen, J. Zheng, and D. Guo, “Analysis ofphenolic compounds in rhubarbs using liquid chromatographycoupled with electrospray ionization mass spectrometry,” Jour-nal of the American Society for Mass Spectrometry, vol. 18, no. 1,pp. 82–91, 2007.

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