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Comparative determination of various vancomycin preparations with positive ionization electrospray LC-MS-MS M. J. Bogusz, H. Hassan and M. Al Tufail Department of Pathology and Laboratory Medicine, King Faisal Specialist Hospital and Research Centre, P.O.Box 3354, MBC 10, 11211 Riyadh, Kingdom of Saudi Arabia
1. Introduction Vancomycin (fig.1) is a glycopeptide antibiotic isolated from both Streptomyces orientalis and Nocardia lucida (1). It was first introduced for treatment of staphylococcal infections in the 1950s and was quickly relegated to the role of alternate drug, due to high incidence of nephrotoxicity and ototoxicity (2). However, the increasing number of Staphylococcus and Streptococcus isolates, resistant to various antibiotics, led to rehabilitation of vancomycin. In addition, new generation of preparations was devoid of most impurities and showed fewer side effects (3). Nevertheless, it was generally believed that drug toxicities could be avoided if serum concentrations were kept below 40 mg/l and therapeutic drug monitoring (TDM) was recommended, with a through level of 5 – 10 mg/l (4, 5). Usually TDM of vancomycin is done with immunoassays (FPIA, EMIT). Recently, also an electrospray LC-MS-MS has been introduced after simple plasma precipitation procedures (6, 7).
Fig.1. The structure of vancomycin (m.w. 1449)
It has been observed in our hospital that the introduction of the new brand of vancomycin was associated with some incidents of nephrotoxicity among patients. The Department of Hospital Pharmacy requested to perform an analytical study concerning various vancomycin preparations which are currently available. The study was performed using LC-MS-MS technique and the results are presented.
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2. Experimental. 2.1. Materials Following vancomycin preparations were analyzed: - Vancomycin Hydrochloride USP Abbott (USA) cont. vancomycin hydrochloride equivalent to 1000 mg base per vial - Vancocin HCl USP Lilly (USA) cont. vancomycin hydrochloride equivalent to 1000 mg base per vial - Vancomycin hydrochloride DUMEX (Denmark) cont. vancomycin hydrochloride equivalent to 500 mg base per vial - Vancomycin hydrochloride for Injection BP Faulding (UK) cont. vancomycin hydrochloride equivalent to 1000 mg base per vial - Vancomycine Merck (Greece) cont. vancomycin hydrochloride equivalent to 500 mg base per vial From each preparation, stock solutions of 1 mg/ml in acetonitrile-water (20:80) were prepared. 2.2. LC-MS-MS Following instrumentation was applied: TSQ Quantum triple quadrupole LC-MS-MS (Thermo Finnigan, USA) with quaternary Surveyor pump and Surveyor AS autosampler. Electrospray ion source in positive mode was used. The conditions (spray voltage, sheath and auxiliary gas flow, tube lens voltage, capillary temperature) were optimized for vancomycin using solution of the drug in the mobile phase (acetonitrile – ammonium formate buffer 10 mM, pH 3.0 (20:80), introduced directly to the instrument using syringe infusion (at 5 µl/min) combined through tee-joint with the mobile phase flow at 200 µl/min. Full scan analysis was done in the range 100 > 1500 amu on the 1st quadrupole, the collision energy was set at 5 v. Chromatographic analysis was performed on Superspher ODS column 125 x 2 mm (Merck). For quantitative MRM analysis, isocratic elution in mobile phase: acetonitrile – ammonium formate buffer 10 mM, pH 3.0 (20:80) at flow rate of 200 µl/min was done. For full-scan profiling of vancomycin preparations, gradient elution was applied, using following profile: 20% acetonitrile in ammonium formate buffer for 1 min, raise to 50% acetonitrile in 5 min, hold 50% acetonitrile for 5 min. The flow rate was 200 µl//min. 3. Results. Full-scan MS analysis showed the ion m/z 725 amu as the most intensive in all vancomycin preparations (fig.2). This ion corresponds to doubly charged ion of vancomycin. Such finding is quite common for peptide molecules with mass between 1000 and 2000 amu. Small ion m/z 1450 (single-protonated molecular ion) was also visible. Product scan analysis of m/z 725, performed in the range m/z 100 > 1500 amu in infused vancomycin solution, revealed two main product ions: m/z 1306 and 144 (Fig 3). These transitions have been used for MRM analysis of vancomycin preparations. MRM analysis was done from vancomycin solutions containing 1 ng/µl, the injection volume was 5 µl. The peak of vancomycine was observed at Rt around 1.9 min. Signal intensities for all preparations were similar (Fig. 4 a -4 e).
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Abbottfullscan #169 RT: 2.97 AV: 1 SB: 80 0.90-2.28 NL: 8.77E5 T: + c sid=-10.00 Q1MS [ 100.00-1500.00] 724.8 100 95
[M+2H]2+
90 85 80 75 70
VANCOMYCIN
Relative Abundance
65 60
725.8
55 50 45 40 35 30 25
[M+H]+
20 15 10 5
143.6 1459.1
766.5
198.2 231.1 271.3 350.5 412.8
553.7 717.2 625.8
825.4 857.1
966.6
1126.9 1197.7
1306.3
1450.0
0 200
400
600
800 m/z
1000
1200
1400
Fig.2. Mass spectrum of vancomycin. Full-scan (100 > 1500 amu) on 1st quadrupole.
Abbottprods can #87-93 RT: 1.42-1.52 AV: 7 NL: 2.24E6 T: + p s id=-5.00 Full m s 2
[email protected] [ 100.00-1500.00] 724.8
100 95 90 85 80 75 70
RelativeAbundance
65 60 1306.5
55 50
Vancom ycin Product s can of m /z 725
143.8
45 40 35 30 25 20 15 1144.5
10 5
1116.6 200.3 271.1
404.7
0 200
400
769.4 438.5
571.8 600
650.5 800 m /z
855.0
925.0
1044.1 1000
Fig.3. Product scan mass spectrum of m/z 725.
1289.4 1260.3 1200
1332.7 1432.5 1388.0 1464.2 1400
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RT: 0.00 - 4.00 SM: 15G RT: 0.00 - 4.00 SM: 15G NL: 6.55E4 TIC F: + p sid=-10.00 t SRM ms2
[email protected] [ 142.50-145.50] MS ICIS VancoAbbott2
RT: 1.83 AA: 1466602 SN: 1104
100 90
100
RT: 1.86 AA: 2168610 SN: 1853
NL: 9.67E4
RT: 1.88 AA: 3123610 SN: 1710
NL: 1.38E5 TIC F: + p sid=-10.00 t SRM ms2
[email protected] [ 1306.50-1309.50] MS ICIS VancoLilly2
TIC F: + p sid=-10.00 t SRM ms2
[email protected] [ 142.50-145.50] MS ICIS VancoLilly2
90 80 R e l a ti v e A b u n d a n c e
R e la tiv e A b u n d a n c e
80 70 60 50 40 30
70 60 50 40 30 20
20
10
10
0
0
NL: 9.61E4 TIC F: + p sid=-10.00 t SRM ms2
[email protected] [ 1306.50-1309.50] MS ICIS VancoAbbott2
RT: 1.88 AA: 2133798 SN: 1853
100 90 80
100 90 80
70
70
60
60
50
50
40
40
30
30
20
20
10
10 0
0 0.0
0.5
1.0
1.5
2.0 Time (min)
2.5
3.0
0.0
3.5
A
0.5
1.0
1.5
2.0 Time (min)
2.5
3.0
3.5
B RT: 0.00 - 4.00 SM: 15G
RT: 0.00 - 4.00 SM: 15G NL: 1.02E5
RT: 1.86 AA: 2262912 SN: 2006
100
TIC F: + p sid=-10.00 t SRM ms2
[email protected] [ 142.50-145.50] MS ICIS VancoDumex2
90
100
50 40 30
RT: 1.88 AA: 3824653 SN: 3197
NL: 1.72E5 TIC F: + p sid=-10.00 t SRM ms2
[email protected] [ 1306.50-1309.50] MS ICIS VancoDBL1
80 R e l a ti v e A b u n d a n c e
R e l a ti v e A b u n d a n c e
60
NL: 1.22E5 TIC F: + p sid=-10.00 t SRM ms2
[email protected] [ 142.50-145.50] MS ICIS VancoDBL1
90
80 70
RT: 1.89 AA: 2795258 SN: 2370
70 60 50 40 30 20
20
10
10
0
0
NL: 1.45E5 TIC F: + p sid=-10.00 t SRM ms2
[email protected] [ 1306.50-1309.50] MS ICIS VancoDumex2
RT: 1.88 AA: 3265174 SN: 1659
100 90 80
100 90 80
70
70
60
60
50
50
40
40
30
30
20
20
10
10 0
0 0.0
0.5
C
1.0
1.5
2.0 Time (min)
2.5
3.0
3.5
0.0
0.5
1.0
1.5
2.0 Time (min)
2.5
3.0
3.5
D Fig.4 . Mass chromatograms of vancomycin preparations from Abbott (A), Lilly (B), Dumex (C) and Faulding (D). 5 ng amounts were injected.
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RT: 0.00 - 4.00 SM: 15G
100
RT: 1.89 AA: 3257199 SN: 2187
NL: 1.40E5
RT: 1.88 AA: 4382795 SN: 3056
NL: 1.92E5 TIC F: + p sid=-10.00 t SRM ms2
[email protected] [ 1306.50-1309.50] MS ICIS VancoMerck1
TIC F: + p sid=-10.00 t SRM ms2
[email protected] [ 142.50-145.50] MS ICIS VancoMerck1
90
R e la tiv e A b u n d a n c e
80 70 60 50 40 30 20 10 0
100 90 80 70 60 50 40 30 20 10 0 0.0
0.5
1.0
1.5
2.0 Time (min)
2.5
3.0
3.5
E Fig.4 (continued). Mass chromatograms of vancomycin preparations from Merck (E). 5 ng amounts were injected.
Figs. 5, 6, 7, 8, and 9 show mass chromatograms of vancomycin solutions, analyzed in full scan mode in gradient elution conditions. In all cases very similar chromatograms were observed, without indication for any different peak pattern in particular sample.
RT: 0.00 - 9.99
SM: 15G RT: 9.03 AA: 192455736 SN: 534
100 80 RT: 7.14 AA: 125962362 SN: 585
60 RT: 0.64 AA: 4662120 SN: 37
40 20 0
RT: 4.91 AA: 6626571 SN: 34
RT: 2.39 AA: 3507375 SN: 32
RT: 1.92 AA: 1323971 SN: 698
100 RelativeAbundance
RT: 1.76 AA: 8908675 SN: 65
NL: 4.04E7 TIC MS ICIS VancoAbbott2 _040128112 537
VANCOMYCIN ABBOTT 80 60 RT: 7.80 RT: 7.15 AA: 256633 AA: 233200 SN: 135 SN: 127
40 20 0
RT: 7.66 AA: 190182 SN: 373
100 80
RT: 9.57 AA: 315156 SN: 132
RT: 8.24 AA: 232723 SN: 468
RT: 9.20 AA: 122208 SN: 176
60
NL: 9.42E4 m /z= 724.5-725.5 MS ICIS VancoAbbott2 _040128112 537
NL: 2.93E4 m /z= 1449.51450.5 MS ICIS VancoAbbott2 _040128112 537
40 20 0 0
1
2
3
4
5 Tim e (m in)
6
7
8
9
Fig. 5. Full scan LC-MS analysis of vancomycin Abbott. A: TIC trace, B: mass trace m/z 725, C: mass trace m/z 1450.
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RT: 0.00 - 9.98
SM: 15G NL: 2.67E7
RT: 8.69 AA: 114495956 SN: 442
100 RT: 6.09 AA: 1994155 SN: 27
80 60
RT: 0.48 AA: 9363452 SN: 64
40
RT: 1.74 AA: 15288578 SN: 100
RT: 3.74 AA: 9116225 SN: 54
TIC MS ICIS VancoLilly2_ 0401281156 53
RT: 7.14 AA: 116704711 SN: 559
RT: 4.39 AA: 4334154 SN: 46
20 0
m /z= 724.5-725.5 RT: 9.49 MS ICIS AA: 388904 VancoLilly2_ SN: 191 0401281156 53
80 RT: 7.00 AA: 322324 SN: 187
VANCOMYCIN LILLY 60 40 20
RT: 8.25 AA: 54926 SN: 57
0 RT: 8.02 AA: 103277 SN: 274
100
RT: 9.18 AA: 82181 SN: 291
80 RT: 6.74 AA: 32208 SN: 118
60 40
NL: 1.03E4 m /z= 1449.51450.5 MS ICIS VancoLilly2_ 0401281156 53
20 0 0
1
2
3
4
5 Tim e (m in)
6
7
8
9
Fig. 6. Full scan LC-MS analysis of vancomycin Lillyt. A: TIC trace, B: mass trace m/z 725, C: mass trace m/z 1450.
RT: 0.00 - 9.98
SM: 15G
100 RT: 7.13 AA: 134701639 SN: 586
80 RT: 1.74 RT: 0.46 AA: 3228973 AA: 13261670 SN: 117 SN: 29
60 40
RT: 9.04 AA: 70648103 SN: 254
NL: 2.54E7 TIC MS ICIS VancoDum ex 1_04012812 1227
RT: 5.27 AA: 2738283 SN: 31
RT: 3.23 AA: 5767480 SN: 32
20 0
NL: 6.44E4
RT: 1.88 AA: 1012238 SN: 476
100 RelativeAbundance
RelativeAbundance
NL: 6.63E4
RT: 1.92 AA: 1218622 SN: 436
100
m /z= 724.5-725.5 MS ICIS VancoDum ex RT: 9.79 AA: 158028 1_04012812 1227 SN: 135
VANCOMYCIN DUMEX
80 60
RT: 7.25 AA: 259635 SN: 130
40 20
RT: 7.92 AA: 87910 SN: 47
0
RT: 9.21 AA: 138992 SN: 763
100 RT: 7.92 AA: 93264 SN: 511
80 60
RT: 8.44 AA: 32408 SN: 188
40
NL: 1.69E4 m /z= 1449.51450.5 MS ICIS VancoDum ex 1_04012812 1227
20 0 0
1
2
3
4
5 Tim e (m in)
6
7
8
9
Fig. 7. Full scan LC-MS analysis of vancomycin Dumex. A: TIC trace, B: mass trace m/z 725, C: mass trace m/z 1450.
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RT: 0.00 - 9.99
SM: 15G
100
RT: 7.21 AA: 91347023 SN: 508
80 RT: 1.81 AA: 6777503 SN: 74
60
NL: 2.27E7
RT: 8.93 AA: 37034477 SN: 142
TIC MS ICIS VancoDBL1 _040128124 344
RT: 2.67 AA: 1217354 SN: 20
40 20 0
RelativeAbundance
NL: 7.98E4
RT: 2.02 AA: 1116848 SN: 672
100
m /z= 724.5-725.5 MS ICIS VancoDBL1 _040128124 344
VANCOMYCIN FAULDING 80 60
RT: 7.23 AA: 317514 SN: 172
40
RT: 7.77 AA: 105569 SN: 119
RT: 9.45 AA: 104953 SN: 64
20 0
NL: 8.08E3 RT: 9.01 AA: 46129 SN: 626
100 80
RT: 9.82 AA: 7645 SN: 171
60 40
m /z= 1449.51450.5 MS ICIS VancoDBL1 _040128124 344
20 0 0
1
2
3
4
5 Tim e (m in)
6
7
8
9
Fig. 8. Full scan LC-MS analysis of vancomycin Faulding. A: TIC trace, B: mass trace m/z 725, C: mass trace m/z 1450.
RT: 0.00 - 9.98
SM: 15G RT: 7.23 AA: 144189529 SN: 645
100 80
RT: 1.11 AA: 5389428 SN: 64
60
RT: 1.85 AA: 20658040 SN: 163
RT: 4.27 RT: 2.87 AA: 11556000 AA: 15090307 SN: 99 SN: 97
NL: 1.84E7
RT: 8.69 AA: 49667931 SN: 202
TIC MS ICIS VancoMerck1 _040128131 500
RT: 5.71 AA: 9591352 SN: 64
40 20 0
100 RelativeAbundance
NL: 9.99E4
RT: 2.02 AA: 1422369 SN: 1065 VANCOMYCIN MERCK
80 60
RT: 8.86 AA: 304291 SN: 294
RT: 7.04 AA: 174794 SN: 153
40 20 0
RT: 6.13 AA: 74460 SN: 396
100
RT: 8.76 AA: 49391 SN: 260
80 60
RT: 8.46 AA: 15711 SN: 92
40
m /z= 724.5-725.5 MS ICIS VancoMerck1 _040128131 500
NL: 8.86E3 RT: 9.62 AA: 63455 m /z= SN: 350 1449.51450.5 MS ICIS VancoMerck1 _040128131 500
20 0 0
1
2
3
4
5 Tim e (m in)
6
7
8
9
Fig. 9. Full scan LC-MS analysis of vancomycin Merck. A: TIC trace, B: mass trace m/z 725, C: mass trace m/z 1450.
Generally, the study of five vancomycin preparations did not reveal any differences, detectable in LC-MS and LC-MS-MS analysis. Most probably, observed side effects are not related to any particular brand of this drug.
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References 1. H. R. Perkin. Pharmacol.Ther. 16 (1982) 181-197. 2. J.E. Geraci, F. R. Hellman, D.R.Nichols, W.E.Wellman, G.T.Ross. Antibiotics Annual 1956-1957 (1957) 90-106. 3. B. F. Farber, R. C. Moellering. Antimicrob.Agents Chemother. 23 (1983) 138-141. 4. M. J. Rybak, L. M. Albrecht, S. C. Boike, P. H. Chandrasekar. J. Antimicrob. Ther. 25 (1990) 679-687. 5. C. M. Tobin, J. M. Darville, A. H. Thomson, G. Sweeney, J. F. Wilson, A. P. MacGowan, L. O. White. J. Antimicrob. Ther. 50 (2002) 713-718. 6. R. T. Cass, J. S. Villa, D. E. Karr, D. E. Schmidt. Rapid Commun.Mass Spectrom. 15 (2001) 406-412. 7. N. Shibata, M. Ishida, Y. V. Prasad, W. Gao, Y. Yoshikawa, K. Takada. J. Chromatogr. B 789 (2003) 211218.
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