LAAN-A-LM-E039

Application News
Liquid Chro ma t o g r a p h y Ma ss S p e c tro m e tr y N o . C68

Analysis of Drug Degradants by LC/MS

This Application News introduces an example of high- with in-source CID (Collision Induced Dissociation) to
speed analysis of drug degradants by LC/MS. produce experimental results. In addition, formula
The ultra fast features of the LCMS-2020, scan rates prediction was conducted using the LCMS-IT-TOF, and
up to 15,000 u/sec and positive/negative polarity separation of the sample components was carried out
switching as fast as 15 msec, were used in combination using the Prominence UFLCXR.
■ Analysis of Penicillin G Using the LCMS-2020
Fig. 1 shows a UV chromatogram and TIC chromatogram mass spectra were obtained for one UFLC peak under
of penicillin G (1 mg/mL aqueous solution). We each set of MS conditions. Fragmentation ions of
conducted a total of 6 measurements, all using penicillin G were generated as a result of in-source
different MS conditions consisting of positive or CID. However, reduced sensitivity was seen at the
negative mode in conjunction with a range of 3 high voltage settings of (c) 70 V and (f) -70 V of the Q-
different DL and Q-array DC voltages (Fig. 2). At a array due to the loss of normal ion focusing at these
scan speed of 15,000 u/sec, each mass spectrum was values. Fig. 3 shows the fragmentation ion
acquired within 0.05 sec. A complete cycle of voltage assignments.
and polarity tests took 0.3 sec, and approximately 20
Inten. (x1,000,000)
3.5
1
3.0 1: penicillin G
2.5

2.0
UV 220 nm
1.5

1.0

0.5
MS positive, DL: 0 V,
Q-array DC: 0 V
0.0
0.0 1.0 2.0 3.0 4.0 5.0 min

Fig. 1 Chromatograms of Penicillin G Standard Solution Fig. 3 Fragmentation of Penicillin G

(a) positive, DL: 0 V, Q-array DC: 0 V (d) negative, DL: 0 V, Q-array DC: 0 V
335 333
100 100

160 192
50 373 50
176 289
357 64 113
0 0
100 200 300 400 m/z 100 200 300 400 m/z
(b) positive, DL: 50 V, Q-array DC: 50 V (e) negative, DL: -50 V, Q-array DC: -50 V
160 192
100 100
114 357
50 50 74
87 189 171
70 335 117 215 289 318
69 407 438 475
0 0
100 200 300 400 m/z 100 200 300 400 m/z

(c) positive, DL: 70 V, Q-array DC: 70 V (f) negative, DL: -70 V, Q-array DC: -70 V
114 74
100 91
100

70 117
50 160 357 50 70 157 289 361
241 499
479
344 420
335 389
128 283 417 463
0 0
100 200 300 400 m/z 100 200 300 400 m/z

Fig. 2 Mass Spectra of Penicillin G

Mass Spectra of Penicillin G ((a) positive, DL: 0 V, Q-array DC: 0 V, (b) positive, DL: 50 V, Q-array DC: 50 V,
(c) positive, DL: 70 V, Q-array DC: 70 V, (d) negative, DL: 0 V, Q-array DC: 0 V, (e) negative, DL: -50 V, Q-array DC: -50 V,
(f) negative, DL: -70 V, Q-array DC: -70 V)
No.C68

■ Analysis of Penicillin G Degradants Using LCMS-2020 ■ Formula Prediction Using LCMS-IT-TOF

A penicillin G standard solution (10 mg/mL aqueous We also conducted MS/MS measurement using the
solution) was heated at 60 ˚C for 40 hours to carry out LCMS-IT-TOF in order to determine the likely
decomposition. As shown in the chromatogram of Fig. composition of the degradants. Using both positive
4, the penicillin G peak almost completely disappeared. and negative detection, C15H20N2O3S was the top-
It was observed that the mass spectral patterns of ranked prediction. Benzylpenilloic acids (Fig. 6) are
peaks 1 and 2 are very similar to each other. The known to be penicillin G impurities. Considering the
mass spectra of peak 1 are shown in Fig. 5. The formula prediction results and the existence of the 2
molecular weight is thought to be 308, and the similar degradant peaks, it is supposed that these
fragmentation is different from that of penicillin G, so compounds were present in the degradation sample.
the structure is presumed to be different.

Inten. (x1,000,000)
7.5

2
1
5.0
UV 220 nm

2.5 MS positive,
DL: 0 V,
Q-array DC: 0 V
0.0
0.0 1.0 2.0 3.0 4.0 5.0 min

Fig. 4 Chromatograms of Degradation Products of Penicillin G Fig. 6 Structures of Benzylpenilloic Acids

(a) positive, DL: 0 V, Q-array DC: 0 V (d) negative, DL: 0 V, Q-array DC: 0 V
100 309 100 307

50 50
229 276 421
0 0
100 200 300 400 m/z 100 200 300 400 m/z
(b) positive, DL: 50 V, Q-array DC: 50 V (e) negative, DL: -50 V, Q-array DC: -50 V
100 174 100 307
309
50 128 50 229 495
263 394
59 100 331 363 74 134 192 289 350
99 238 452
0 0
100 200 300 400 m/z 100 200 300 400 m/z

(c) positive, DL: 70 V, Q-array DC: 70 V (f) negative, DL: -70 V, Q-array DC: -70 V
100 100 394
91
128 319
50 50 361 395 494
59 192 229
100 174 230 289 418 69 91 127 160 246 276 335 422 461
0 0
100 200 300 400 m/z 100 200 300 400 m/z

Fig. 5 Mass Spectra of Degradation Product of Penicillin G (peak 1)

Mass Spectra of Degradation Product of Penicillin G (peak 1) ((a) positive, DL: 0 V, Q-array DC: 0 V, (b) positive, DL: 50 V, Q-array DC: 50 V,
(c) positive, DL: 70 V, Q-array DC: 70 V, (d) negative, DL: 0 V, Q-array DC: 0 V, (e) negative, DL: -50 V, Q-array DC: -50 V,
(f) negative, DL: -70 V, Q-array DC: -70 V)

Table 1 Analytical Conditions

Column : Shim-pack XR-ODS II (75 mm L. × 2.0 mm I.D., 2.2 μm) Detection

Mobile Phase : A: 50 mmol/L (ammonium) formate buffer (pH 3.9) PDA
B: 100 mmol/L (ammomium) formate buffer (pH 3.9)/ Wavelength : 220 nm
acetonitrile= 1/1 MS (LCMS-2020)
Time Program : 20 %B (0 min) - 50 %B (5 min) - 20 %B (5.01 min) - STOP (10 min) Probe Voltage : +4.5 kV(ESI-Positive mode),
Flow Rate : 0.8 mL/min -3.5 kV (ESI-Negative mode)
Column Temp. : 40 ˚C Nebulizing Gas Flow : 1.5 L/min
Injection Volume : 0.5 μL Drying Gas Flow : 20.0 L/min
DL Temp. : 300 ˚C
Block Heater Temp. : 450 ˚C
DL, Q-array Voltages : (a) DL: 0 V, Q-array DC: 0 V, (b) DL: 50 V, Q-array DC: 50 V,
(c) DL: 70 V, Q-array DC: 70 V, (d) DL: 0 V, Q-array DC: 0 V,
(e) DL: -50 V, Q-array DC: -50 V, (f) DL: -70 V, Q-array DC: -70 V
Event Time : 0.05 sec
Scan Range : m/z 50 - 500

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