Formation of Metal Adduct Solvent Ions or Ion Clusters and the Influence on LC/MS Analysis
Zhe-ming Gu, Dawei Zhou, Xian-guo Zhao, and Jinn Wu
XenoBiotic Laboratories, Inc. 107 Morgan Lane, Plainsboro, New Jersey 08536
Introduction
Results & Discussion
2e4
Metal adduct ions and protonated ions
formed during LC/MS analysis are widely used as
an adjunct for determination of molecular weights
or characterization of molecular structures. If
needed, trace metal ions, e.g., Na+ or Li+, are
introduced to enhance intensity and consistency of
adduct ions or to form desired adducts. The metal
ions added, however, also generate relatively stable
metal adduct solvent ions in various combinations.
The adduct ions are distributed over the entire mass
monitoring range thus making it very difficult to
determine the molecular ions of unknown chemical
entities. In the multiple reaction monitoring mode,
the presence of adduct ions with similar mass units
to the molecular ion of an analyte may also
significantly increase instrument background.
Experiments were designed and conducted to
investigate the formation of sodium adduct solvent
ions or ion clusters
Isocratic:
Flow:
1.4e6
PE Sciex API-365
Ion Spray:
Turbo Gas Temp:
Orifice:
Ring:
Collision Energy:
1.2e4
Na4F5
Without Na
Na5F6 Na6F7
212.4
385.2 453.3
Na5A4
Na6A5
351.2
433.4
Na4A5
*
Na8F9
Na9F10
250
300
400
250
350
450
550
Na8A7
Na9A8
597.0
679.2
Na5A6
469.3
Na7A8
633.0
*
*
500
637.0 719.1 787.0
600 M/Z, amu
869.2
345.0
923.0
800
1000
4e5
100
8e4
427.3 495.3 563.1
300
159.1
645.3
500
187.3
*
600
700
Figure 2. LC/(±)ESI-MS spectra of 25 mM of NH4OAc with
0.5 mM of NaOAc; *Peaks due to the existence of trace
HCOOH in the system (Figures 3 & 4); A: Acetate ion
(CH3COO-) with 59 Da
198.4
The
14
amu
difference
corresponded
to
the
mass
distinction between A and F. The
MS/MS product ion scans revealed
that the ion clusters were formed
by combination of various
numbers of Na+, HCOO- (F), and
CH3COO- (A) (Table I). The peak
intensities could be estimated
statistically
with
binomial
coefficients.
• In the LC/MS/MS quantitation
analysis, part of the background
comes from similar ion transition
of the solvent adducts and an
analyte. NH4OAc with a trace of
Na+ and HCOOH mobile phase,
therefore, would possibly cause
high background.
305.3
241.3 261.4
150
23.2
291.1
249.1
405.3 419.2433.3
391.2
337.3 351.1
323.3
M/Z,amu
350
250
2e4
4e4
269.4
427.3
413.3 441.2
373.2
399.0
91.0
+ Product Ion of m/z 91
1.2e4
317.2
450
B
795.2
M/Z, amu
1000
359.0
255.2
214.4
713.1
700
345.0
263.0
277.2
650
M/Z, amu
Figure 1. LC/(-)ESI-MS spectra of 25 mM of HCOONH4 w/o 0.5
mM of NaOAc; F: formate ion (HCOO-) with 45 Da
Na7A6
M/Z, amu
400
173.3
515.2
*
200
263.0
569.1
331.0
Na6A7
*
LC/(-)ESI-MS of 25 mM NH4OAc+0.01%
HCOOH, 0.5mM NaOAc
212.1
2e5
521.2
2e5
551.0
2e6
4e5
501.2
349.4 419.2
657.0
Negative Mode
387.1
A
269.4
2e5
Na7F8
4e3
305.1
Na3A4
of 25 mM NH4OAc+0.01%
HCOOH, 0.5 mM NaOAc
589.1
LC/ESI-MS of 25 mM
Positive Mode
NH4OAc with 0.5 mM Na
6e6
5e6
LC/ESI-MS and MS/MS in
positive or negative mode
5000 V
400 °C
40 Units
300 Units
15 ~ 40 eV
269.2
Na4A3
Mass Spectrometer Condition
Mode:
Na3F4
158.3 LC/(+)ESI-MS
With 0.5 mM Na
• When 0.01% HCOOH was added to 25 mM NH4OAc containing 0.5 mM NaOAc
solution, ion clusters rather than single ion peaks were observed (Figures 3,4). Each ion
cluster consisted of 3-4 ion peaks with a 14 amu difference between two adjacent peaks.
MS/MS product ion scans revealed that the ion clusters were formed by various
numerical combinations of Na+, HCOO- (F), and CH3COO- (A).
Waters 2690 Separations Module
MS System:
317.2
• Stable sodium adduct solvent clusters from NH4OAc solution were observed in both
positive and negative modes (Figure 2). The MS peaks were revealed to be NanA(n+1)
(A = CH3COO-) for negative mode, and Na(n+1)An for positive mode.
HPLC Condition
CH3CN
25 mM NH4OAc, or NH4OOCH, or
NH4OAc with 1% of HCOOH, with or
without 0.5 mM NaOAc in H2O.
A : B = 50 : 50
0.3 mL/min
249.2
• Stable sodium adduct solvent clusters from HCOONH4 solution were observed in
negative ion mode (Figure 1). The MS peaks were revealed to be NanF(n+1)
(F = HCOO-) by MS/MS product ion scan.
Methods
LC System:
Mobile Phase
A:
B:
4e5
LC/(-)ESI-MS of 25 mM HCOONH4
Solvent
ions
with
molecular weights over 250
Da, e.g., multimer ions of
water, can be detected only
under modest ion source
conditions [1].
Thus,
interference from these
solvent ions is barely
observed during LC/MS
analysis when high source
temperature and high drying
gas flow rate are employed.
The sodium adduct solvent
ions, however, are relatively
stable.
220
300
400
495.3
481.4 509.3
563.1
577.1
591.0
523.0 549.2
500 M/Z, amu
600
Figure 4. LC/(-)ESI-MS spectra of 25 mM of NH4OAc and 0.01% HCOOH with
0.5 mM of NaOAc.
68 (Na +F)
45.1
4e3
20
7e4
23.1
55.2
50
63.0
Positive Mode
72.9
M/Z,amu
100
+ Product Ion of m/z 105
4e4
82 (Na +A)
105.1
64.0
20
60
1.5e5
90.8
2.5e5
95.1
Na6A3F2
240.8 268.8
82
322.9
336.9
Na6A4F
1e5
186.8
172.9
336.9
323.1 350.6
68
1.8e5 +Product Ion of m/z 433
419.1
268.9
Na6A5
432.9
187.0
100
82
200
255.1
82
M/Z, amu
300
350.7
82
400
Figure 3. A: LC/(+)ESI-MS spectra of 25 mM of NH4OAc and 0.01% of HCOOH w/o
0.5 mM of NaOAc; B: The product ion scans of m/z 91, 105, 405, 419, and 433.
References
Na 2A
337
159
Na 3F2
Na 3AF
351
391
Na 5A 4
Na 6A 2F3
m/z
249
Cluster
Na 3F4
373
263
Na 3AF 3
m/z
Cluster
Na 4A 4F
Na 5AF 5
399
277
Na 3A 2F2
413
291
Na 3A 3F
427
Na 5A 2F4
Na 5A 3F3
Na 3A 2
Na 4AF 2
405
Na 6A 3F2
317
Na 5A 4F2
419
Na 6A 4F
331
Na 4F5
Na 4AF 4
441
241
467
255
Na 4A 2F
433
Na 4A 2F3
481
Na 6AF 6
Na 6A 2F5
Na 4A 3
Na 6A 5
Na 7A 2 F4
345
501
359
Na 4A 3F2
495
Na 6A 3F4
269
Conclusion
68
1e5
80
105
Negative Mode
Cluster
Na 5A 2F2
Na 5A 3F
Table I. Peak assignments of the LC/(±)ESI-MS signals of 25 mM NH4OAc and
0.01% HCOOH with 0.5 mM NaOAc (Figures 3 & 4); F: Formate ion with 45 Da;
A: Acetate ion with 59 Da
404.9
82
254.9
268.9
+Product Ion of m/z 419
m/z
323
187
100
M/Z,amu
255.0
172.9
187.1 82
82
Cluster
Na 2F
173
77.0
3.5e5 +Product Ion of m/z 405
m/z
91
• Formation of sodium adduct solvent clusters in different HPLC
mobile phases were extensively analyzed by LC/(±)ESI-MS and
MS/MS. The results indicate that 25 mM NH4OAc buffer with trace
of Na+ ion gives very complicated ion clusters over a wide mass
range. If Na+ addition is necessary, selection of HPLC mobile phases
should be given carefully attention, so that the interference from the
solvent ion clusters can be avoided.
• Similar results were also observed when other metal salts, e.g.,
KCl, LiCl, KCl, and AgNO3, were added to the HPLC aqueous
mobile phase consisting of 25 mM NH4OAc and CH3CN.
1. Ledman, D. W., Fox, R. O., J Am Soc Mass Spectrom 1997, 8, 1158-1164.